Multiple fusion antigens for use in immunoassays for anti-HCV antibodies

ABSTRACT

HCV immunoassays comprising an NS3/4a conformational epitope and a multiple epitope fusion antigen are provided, as well as immunoassay solid supports for use with the immunoassays.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of Ser. No. 09/881,654, filed Jun. 14,2001, now U.S. Pat. No. 6,632,601 B2, which is related to provisionalpatent application Ser. No. 60/212,082, filed Jun. 15, 2000, nowabandoned; 60/280,811, filed Apr. 2, 2001, now abandoned; and60/280,867, flied April 2, 2001, now abandoned, from which applicationspriority is claimed under 35 USC § 119(e)(1) and which applications areincorporated herein by reference in their entireties.

TECHNICAL FIELD

The present invention pertains generally to viral diagnostics. Inparticular, the invention relates to immunoassays using multiple HCVantigens, for accurately diagnosing hepatitis C virus infection.

BACKGROUND OF THE INVENTION

Hepatitis C Virus (HCV) is the principal cause of parenteral non-A,non-B hepatitis (NANBH) which is transmitted largely through body bloodtransfusion and body fluid exchange. The virus is present in 0.4 to 2.0%of the general population in the United States. Chronic hepatitisdevelops in about 50% of infections and of these, approximately 20% ofinfected individuals develop liver cirrhosis which sometimes leads tohepatocellular carcinoma. Accordingly, the study and control of thedisease is of medical importance.

HCV was first identified and characterized as a cause of NANBH byHoughten et al. The viral genomic sequence of HCV is known, as aremethods for obtaining the sequence. See, e.g., International PublicationNos. WO 89/04669; WO 90/11089; and WO 90/14436. HCV has a 9.5 kbpositive-sense, single-stranded RNA genome and is a member of theFlaviridae family of viruses. At least six distinct, but relatedgenotypes of HCV, based on phylogenetic analyses, have been identified(Simmonds et al., J. Gen. Virol. (1993) 74:2391-2399). The virus encodesa single polyprotein having more than 3000 amino acid residues (Choo etal., Science (1989) 244:359-362; Choo et al., Proc. Natl. Acad. Sci. USA(1991) 88:2451-2455; Han et al., Proc. Natl. Acad. Sci. USA (1991)88:1711-1715). The polyprotein is processed co- and post-translationallyinto both structural and non-structural (NS) proteins.

In particular, as shown in FIG. 1, several proteins are encoded by theHCV genome. The order and nomenclature of the cleavage products of theHCV polyprotein is as follows:NH₂-C-E1-E2-P7-NS2-NS3-NS4a-NS4b-NS5a-NS5b-COOH. Initial cleavage of thepolyprotein is catalyzed by host proteases which liberate threestructural proteins, the N-terminal nucleocapsid protein (termed “core”)and two envelope glycoproteins, “E1” (also known as E) and “E2” (alsoknown as E2/NS1), as well as nonstructural (NS) proteins that containthe viral enzymes. The NS regions are termed NS2, NS3, NS4, NS4a, NS4b,NS5a and NS5b. NS2 is an integral membrane protein with proteolyticactivity. NS2, either alone or in combination with NS3, cleaves theNS2-NS3 sissle bond which in turn generates the NS3 N-terminus andreleases a large polyprotein that includes both serine protease and RNAhelicase activities. The NS3 protease serves to process the remainingpolyprotein. Completion of polyprotein maturation is initiated byautocatalytic cleavage at the NS3-NS4a junction, catalyzed by the NS3serine protease. Subsequent NS3-mediated cleavages of the HCVpolyprotein appear to involve recognition of polyprotein cleavagejunctions by an NS3 molecule of another polypeptide. In these reactions,NS3 liberates an NS3 cofactor (NS4a), NS4b and NS5a (NS5A has aphosphorylation function), and an RNA-dependent RNA polymerase (NS5b).

A number of general and specific polypeptides useful as immunologicaland diagnostic reagents for HCV, derived from the HCV polyprotein, havebeen described. See, e.g., Houghton et al., European Publication Nos.318,216 and 388,232; Choo et al., Science (1989) 244:359-362; Kuo etal., Science (1989) 244:362-364; Houghton et al., Hepatology (1991)14:381-388; Chien et al., Proc. Natl. Acad. Sci. USA (1992)89:10011-10015; Chien et al., J. Gastroent. Hepatol. (1993) 8:S33-39;Chien et al., International Publication No. WO 93/00365; Chien, D. Y.,International Publication No. WO 94/01778. These publications provide anextensive background on HCV generally, as well as on the manufacture anduses of HCV polypeptide immunological reagents. For brevity, therefore,the disclosure of these publications is incorporated herein byreference.

Sensitive, specific methods for screening and identifying carriers ofHCV and HCV-contaminated blood or blood products would provide animportant advance in medicine. Post-transfusion hepatitis (PTH) occursin approximately 10% of transfused patients, and HCV has accounted forup to 90% of these cases. Patient care as well as the prevention andtransmission of HCV by blood and blood products or by close personalcontact require reliable diagnostic and prognostic tools. Accordingly,several assays have been developed for the serodiagnosis of HCVinfection. See, e.g., Choo et al., Science (1989) 244:359-362; Kuo etal., Science (1989) 244:362-364; Choo et al., Br. Med. Bull. (1990)46:423-441; Ebeling et al., Lancet (1990) 335:982-983; van der Poel etal., Lancet (1990) 335:558-560; van der Poel et al., Lancet (1991)337:317-319; Chien, D. Y., International Publication No. WO 94/01778;Valenzuela et al., International Publication No. WO 97/44469; andKashiwakuma et al., U.S. Pat. No. 5,871,904.

A significant problem encountered with some serum-based assays is thatthere is a significant gap between infection and detection of the virus,often exceeding 80 days. This assay gap may create great risk for bloodtransfusion recipients. To overcome this problem, nucleic acid-basedtests (NAT) that detect viral RNA directly, and HCV core antigen teststhat assay viral antigen instead of antibody response, have beendeveloped. See, e.g., Kashiwakuma et al., U.S. Pat. No. 5,871,904.

However, there remains a need for sensitive, accurate diagnostic andprognostic tools in order to provide adequate patient care as well as toprevent transmission of HCV by blood and blood products or by closepersonal contact.

SUMMARY OF THE INVENTION

The present invention is based in part, on the finding that the use ofNS3/4a conformational epitopes, in combination with multiple epitopefusion antigens, provides a sensitive and reliable method for detectingearly HCV seroconversion. The assays described herein can also detectHCV infection caused by any of the six known genotypes of HCV. The useof multiple epitope fusion proteins also has the added advantages ofdecreasing masking problems, improving sensitivity in detectingantibodies by allowing a greater number of epitopes on a unit area ofsubstrate, and improving selectivity.

Accordingly, in one embodiment, the subject invention is directed to animmunoassay solid support consisting essentially of at least one HCVNS3/4a conformational epitope and a multiple epitope fusion antigen,bound thereto, wherein said NS3/4a epitope and/or said multiple epitopefusion antigen react specifically with anti-HCV antibodies present in abiological sample from an HCV-infected individual.

The NS3/4a epitope may comprise the amino acid sequence depicted inFIGS. 3A-3D, or an amino acid sequence with at least 80% sequenceidentity thereto, or 90% sequence identity thereto, or at least 98%sequence identity thereto, or any integer in between, so long as thesequence has protease activity. In certain embodiments, the NS3/4aconformational epitope consists of the amino acid sequence depicted inFIGS. 3A-3D.

In additional embodiments, the multiple epitope fusion antigen comprisesthe amino acid sequence depicted in FIGS. 5A-5F, or an amino acidsequence with at least 80% sequence identity thereto, or 90% sequenceidentity thereto, or at least 98% sequence identity thereto, or anyinteger in between, so long as the sequence reacts specifically withanti-HCV antibodies present in a biological sample from an HCV-infectedindividual. In certain embodiments, the multiple epitope fusion antigenconsists of the amino acid sequence depicted in FIGS. 5A-5F.

In yet another embodiment, the subject invention is directed to animmunoassay solid support consisting essentially of at least one HCVNS3/4a conformational epitope and a multiple epitope fusion antigen,bound thereto, wherein said NS3/4a conformational epitope comprises theamino acid sequence depicted in FIGS. 3A-3D, or an amino acid sequencewith at least 80% sequence identity thereto which has protease activity,and said multiple epitope fusion antigen comprises the amino acidsequence depicted in FIGS. 5A-5F, or an amino acid sequence with atleast 80% sequence identity thereto which reacts specifically withanti-HCV antibodies present in a biological sample from an HCV-infectedindividual. In certain embodiments, the NS3/4a conformational epitopeand the multiple epitope fusion antigen have at least 90%, 98% (or anyinteger between) sequence identity to the amino acid sequences of FIGS.3A-3D and FIGS. 5A-5F, respectively, so long as the NS3/4a sequence hasprotease activity, and the multiple epitope fusion antigen reactsspecifically with anti-HCV antibodies present in a biological samplefrom an HCV-infected individual. In certain embodiments, the NS3/4aconformational epitope consists of the amino acid sequence depicted inFIGS. 3A-3D, and the multiple epitope fusion antigen consists of theamino acid sequence depicted in FIGS. 5A-5F.

In another embodiment, the invention is directed to an immunoassay solidsupport consisting essentially of at least one HCV NS3/4a conformationalepitope and a multiple epitope fusion antigen, bound thereto, whereinsaid NS3/4a conformational epitope consists of the amino acid sequencedepicted in FIGS. 3A-3D, and said multiple epitope fusion antigenconsists of the amino acid sequence depicted in FIGS. 5A-5F.

In still a further embodiment, the invention is directed to a method ofdetecting hepatitis C virus (HCV) infection in a biological sample, saidmethod comprising:

(a) providing an immunoassay solid support as described above;

(b) combining a biological sample with said solid support underconditions which allow HCV antibodies, when present in the biologicalsample, to bind to said NS3/4a epitope and/or said multiple epitopefusion antigen to form a first immune complex;

(c) adding to the solid support from step (b) under complex formingconditions a detectably labeled antibody, wherein said labeled antibodyis reactive with said immune complex;

(d) detecting second immune complexes formed between the detectablylabeled antibody and the first immune complex, if any, as an indicationof HCV infection in the biological sample.

In still a further embodiment, the invention is directed to a method ofdetecting hepatitis C virus (HCV) infection in a biological sample, saidmethod comprising:

(a) providing an immunoassay solid support consisting essentially of atleast one HCV NS3/4a conformational epitope and a multiple epitopefusion antigen, bound thereto, wherein said NS3/4a conformationalepitope consists of the amino-acid sequence depicted in FIGS. 3A-3D, andsaid multiple epitope fusion antigen consists of the amino acid sequencedepicted in FIGS. 5A-5F;

(b) combining a biological sample with said solid support underconditions which allow HCV antibodies, when present in the biologicalsample, to bind to said NS3/4a epitope and/or said multiple epitopefusion antigen to form a first immune complex;

(c) adding to the solid support from step (b) under complex formingconditions a detectably labeled antibody, wherein said labeled antibodyis reactive with said immune complex;

(d) detecting second immune complexes formed between the detectablylabeled antibody and the first immune complex, if any, as an indicationof HCV infection in the biological sample.

In another embodiment, the invention is directed to an immunodiagnostictest kit comprising an immunoassay solid support as described above, andinstructions for conducting the immunodiagnostic test.

In another embodiment, the subject invention is directed to a method ofproducing an immunoassay solid support, comprising:

(a) providing a solid support; and

(b) binding to the solid support at least one HCV NS3/4a conformationalepitope and a multiple epitope fusion antigen, wherein said NS3/4aepitope and/or said multiple epitope fusion antigen react specificallywith anti-HCV antibodies present in a biological sample from anHCV-infected individual.

In certain embodiments, the conformational epitope comprises the aminoacid sequence depicted in FIGS. 3A-3D, or an amino acid sequence with atleast 80% sequence identity thereto, or 90% sequence identity thereto,or at least 98% sequence identity thereto, or any integer in between, solong as the sequence has protease activity; and the multiple epitopefusion antigen comprises the amino acid sequence depicted in FIGS.5A-5F, or an amino acid sequence with at least 80% sequence identitythereto, or 90% sequence identity thereto, or at least 98% sequenceidentity thereto, or any integer in between, so long as the sequencereacts specifically with anti-HCV antibodies present in a biologicalsample from an HCV-infected individual.

In still further embodiments, the NS3/4a conformational epitope consistsof the amino acid sequence depicted in FIGS. 3A-3D and the multipleepitope fusion antigen consists of the amino acid sequence depicted inFIGS. 5A-5F.

In another embodiment, the invention is directed to a method ofproducing an immunoassay solid support, comprising:

(a) providing a solid support; and

(b) binding to the solid support at least one HCV NS3/4a conformationalepitope and a multiple epitope fusion antigen, wherein said NS3/4aconformational epitope consists of the amino acid sequence depicted inFIGS. 3A-3D, and said multiple epitope fusion antigen consists of theamino acid sequence depicted in FIGS. 5A-5F.

In still a further embodiment, the subject invention is directed to amultiple epitope fusion antigen comprising the amino acid sequencedepicted in FIGS. 5A-5F, or an amino acid sequence with at least 80%sequence identity thereto, or 90% sequence identity thereto, or an aminoacid sequence with at least 98% sequence identity thereto, or anyinteger in between, which sequence reacts specifically with anti-HCVantibodies present in a biological sample from an HCV-infectedindividual.

In certain embodiments, the multiple epitope fusion antigen consists ofthe amino acid sequence depicted in FIGS. 5A-5F.

In other embodiments, the invention is directed to a polynucleotidecomprising a coding sequence for the multiple epitope fusion antigen, arecombinant vector comprising the polynucleotide and control elementsoperably linked to said polynucleotide whereby the coding sequence canbe transcribed and translated in a host cell, a host cell transformedwith the recombinant vector, and a method of producing a recombinantmultiple epitope fusion antigen comprising providing a population ofhost cells as above and culturing said population of cells underconditions whereby the multiple epitope fusion antigen encoded by thecoding sequence present in said recombinant vector is expressed.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. In addition, various references are set forth herein whichdescribe in more detail certain procedures or compositions, and aretherefore incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic representation of the HCV genome, depicting thevarious regions of the polyprotein from which the present assay reagents(proteins and antibodies) are derived.

FIG. 2 is a schematic drawing of a representative immunoassay under theinvention.

FIGS. 3A through 3D depict the DNA (SEQ ID NO:1) and corresponding aminoacid (SEQ ID NO:2) sequence of a representative NS3/4a conformationalantigen for use in the present assays. The amino acids at positions 403and 404 of FIGS. 3A through 3D represent substitutions of Pro for Thr,and Ile for Ser, of the native amino acid sequence of HCV-1.

FIG. 4 is a diagrammatic representation of MEFA 7.1.

FIGS. 5A-5F depict the DNA (SEQ ID NO:3) and corresponding amino acid(SEQ ID NO:4) sequence of MEFA 7.1.

FIGS. 6A-6C show representative MEFAs for use with the subjectimmunoassays. FIG. 6A is a diagrammatic representation of MEFA 3. FIG.6B is a diagrammatic representation of MEFA 5. FIG. 6C is a diagrammaticrepresentation of MEFA 6.

FIGS. 7A-7D are diagrams of the construction of psMEFA7.

FIG. 8 is a diagram of the construction of psMEFA7.1.

FIG. 9 is a diagram of the construction of pd.HCV1a.ns3ns4aPI.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, recombinantDNA techniques and immunology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., FundamentalVirology, 2nd Edition, vol. I & II (B. N. Fields and D. M. Knipe, eds.);Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C.Blackwell eds., Blackwell Scientific Publications); T. E. Creighton,Proteins: Structures and Molecular Properties (W. H. Freeman andCompany, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc.,current addition); Sambrook, et al., Molecular Cloning: A LaboratoryManual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N.Kaplan eds., Academic Press, Inc.).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to “an antigen” includes a mixture of two or more antigens,and the like.

The following amino acid abbreviations are used throughout the text:

Alanine: Ala (A) Arginine: Arg (R) Asparagine: Asn (N) Aspartic acid:Asp (D) Cysteine: Cys (C) Glutamine: Gln (Q) Glutamic acid: Glu (E)Glycine: Gly (G) Histidine: His (H) Isoleucine: Ile (I) Leucine: Leu (L)Lysine: Lys (K) Methionine: Met (M) Phenylalanine: Phe (F) Proline: Pro(P) Serine: Ser (S) Threonine: Thr (T) Tryptophan: Trp (W) Tyrosine: Tyr(Y) Valine: Val (V)

I. Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

The terms “polypeptide” and “protein” refer to a polymer of amino acidresidues and are not limited to a minimum length of the product. Thus,peptides, oligopeptides, dimers, multimers, and the like, are includedwithin the definition. Both full-length proteins and fragments thereofare encompassed by the definition. The terms also include postexpressionmodifications of the polypeptide, for example, glycosylation,acetylation, phosphorylation and the like. Furthermore, for purposes ofthe present invention, a “polypeptide” refers to a protein whichincludes modifications, such as deletions, additions and substitutions(generally conservative in nature), to the native sequence, so long asthe protein maintains the desired activity. These modifications may bedeliberate, as through site-directed mutagenesis, or may be accidental,such as through mutations of hosts which produce the proteins or errorsdue to PCR amplification.

An HCV polypeptide is a polypeptide, as defined above, derived from theHCV polyprotein. The polypeptide need not be physically derived fromHCV, but may be synthetically or recombinantly produced. Moreover, thepolypeptide may be derived from any of the various HCV strains andisolates, such as, but not limited to, any of the isolates from strains1, 2, 3, 4, 5 or 6 of HCV. A number of conserved and variable regionsare known between these strains and, in general, the amino acidsequences of epitopes derived from these regions will have a high degreeof sequence homology, e.g., amino acid sequence homology of more than30%, preferably more than 40%, when the two sequences are aligned. Thus,for example, the term “NS3/4a” polypeptide refers to native NS3/4a fromany of the various HCV strains, as well as NS3/4a analogs, muteins andimmunogenic fragments, as defined further below. The complete genotypesof many of these strains are known. See, e.g., U.S. Pat. No. 6,150,087and GenBank Accession Nos. AJ238800 and AJ238799.

The terms “analog” and “mutein” refer to biologically active derivativesof the reference molecule, or fragments of such derivatives, that retaindesired activity, such as immunoreactivity in the assays describedherein. In general, the term “analog” refers to compounds having anative polypeptide sequence and structure with one or more amino acidadditions, substitutions (generally conservative in nature) and/ordeletions, relative to the native molecule, so long as the modificationsdo not destroy immunogenic activity. The term “mutein” refers topeptides having one or more peptide mimics (“peptoids”), such as thosedescribed in International Publication No. WO 91/04282. Preferably, theanalog or mutein has at least the same immunoactivity as the nativemolecule. Methods for making polypeptide analogs and muteins are knownin the art and are described further below.

Particularly preferred analogs include substitutions that areconservative in nature, i.e., those substitutions that take place withina family of amino acids that are related in their side chains.Specifically, amino acids are generally divided into four families: (1)acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine;(3) non-polar—alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine,asparagine, glutamine, cysteine, serine threonine, tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified asaromatic amino acids. For example, it is reasonably predictable that anisolated replacement of leucine with isoleucine or valine, an aspartatewith a glutamate, a threonine with a serine, or a similar conservativereplacement of an amino acid with a structurally related amino acid,will not have a major effect on the biological activity. For example,the polypeptide of interest may include up to about 5-10 conservative ornon-conservative amino acid substitutions, or even up to about 15-25conservative or non-conservative amino acid substitutions, or anyinteger between 5-25, so long as the desired function of the moleculeremains intact. One of skill in the art may readily determine regions ofthe molecule of interest that can tolerate change by reference toHopp/Woods and Kyte-Doolittle plots, well known in the art.

By “fragment” is intended a polypeptide consisting of only a part of theintact full-length polypeptide sequence and structure. The fragment caninclude a C-terminal deletion and/or an N-terminal deletion of thenative polypeptide. An “immunogenic fragment” of a particular HCVprotein will generally include at least about 5-10 contiguous amino acidresidues of the full-length molecule, preferably at least about 15-25contiguous amino acid residues of the full-length molecule, and mostpreferably at least about 20-50 or more contiguous amino acid residuesof the full-length molecule, that define an epitope, or any integerbetween 5 amino acids and the full-length sequence, provided that thefragment in question retains immunoreactivity in the assays describedherein. For example, preferred immunogenic fragments, include but arenot limited to fragments of HCV core that comprise, e.g., amino acids10-45, 10-53, 67-88, and 120-130 of the polyprotein, epitope 5-1-1 (inthe NS4a/NS4b region of the viral genome) as well as defined epitopesderived from any of the regions of the polyprotein shown in FIG. 1, suchas but not limited to the E1, E2, NS3 (e.g., polypeptide c33c from theNS3 region), NS4 (e.g., polypeptide c100 from the NS3/NS4 regions),NS3/4a and NS5 regions of the HCV polyprotein, as well as any of theother various epitopes identified from the HCV polyprotein. See, e.g.,Chien et al., Proc. Natl. Acad. Sci. USA (1992) 89:10011-10015; Chien etal., J. Gastroent. Hepatol. (1993) 8:S33-39; Chien et al., InternationalPublication No. WO 93/00365; Chien, D. Y., International Publication No.WO 94/01778; U.S. Pat. Nos. 6,150,087 and 6,121,020, all of which areincorporated by reference herein in their entireties.

The term “epitope” as used herein refers to a sequence of at least about3 to 5, preferably about 5 to 10 or 15, and not more than about 1,000amino acids (or any integer therebetween), which define a sequence thatby itself or as part of a larger sequence, binds to an antibodygenerated in response to such sequence. There is no critical upper limitto the length of the fragment, which may comprise nearly the full-lengthof the protein sequence, or even a fusion protein comprising two or moreepitopes from the HCV polyprotein. An epitope for use in the subjectinvention is not limited to a polypeptide having the exact sequence ofthe portion of the parent protein from which it is derived. Indeed,viral genomes are in a state of constant flux and contain severalvariable domains which exhibit relatively high degrees of variabilitybetween isolates. Thus the term “epitope” encompasses sequencesidentical to the native sequence, as well as modifications to the nativesequence, such as deletions, additions and substitutions (generallyconservative in nature).

Regions of a given polypeptide that include an epitope can be identifiedusing any number of epitope mapping techniques, well known in the art.See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology,Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J. Forexample, linear epitopes may be determined by e.g., concurrentlysynthesizing large numbers of peptides on solid supports, the peptidescorresponding to portions of the protein molecule, and reacting thepeptides with antibodies while the peptides are still attached to thesupports. Such techniques are known in the art and described in, e.g.,U.S. Pat. No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA81:3998-4002; Geysen et al. (1985) Proc. Natl. Acad. Sci. USA82:178-182; Geysen et al. (1986) Molec. Immunol. 23:709-715, allincorporated herein by reference in their entireties. Using suchtechniques, a number of epitopes of HCV have been identified. See, e.g.,Chien et al., Viral Hepatitis and Liver Disease (1994) pp. 320-324, andfurther below. Similarly, conformational epitopes are readily identifiedby determining spatial conformation of amino acids such as by, e.g.,x-ray crystallography and 2-dimensional nuclear magnetic resonance. See,e.g., Epitope Mapping Protocols, supra. Antigenic regions of proteinscan also be identified using standard antigenicity and hydropathy plots,such as those calculated using, e.g., the Omiga version 1.0 softwareprogram available from the Oxford Molecular Group. This computer programemploys the Hopp/Woods method, Hopp et al., Proc. Natl. Acad. Sci USA(1981) 78:3824-3828 for determining antigenicity profiles, and theKyte-Doolittle technique, Kyte et al., J. Mol. Biol. (1982) 157:105-132for hydropathy plots.

As used herein, the term “conformational epitope” refers to a portion ofa full-length protein, or an analog or mutein thereof, having structuralfeatures native to the amino acid sequence encoding the epitope withinthe full-length natural protein. Native structural features include, butare not limited to, glycosylation and three dimensional structure. Thelength of the epitope defining sequence can be subject to widevariations as these epitopes are believed to be formed by thethree-dimensional shape of the antigen (e.g., folding). Thus, aminoacids defining the epitope can be relatively few in number, but widelydispersed along the length of the molecule, being brought into correctepitope conformation via folding. The portions of the antigen betweenthe residues defining the epitope may not be critical to theconformational structure of the epitope. For example, deletion orsubstitution of these intervening sequences may not affect theconformational epitope provided sequences critical to epitopeconformation are maintained (e.g., cysteines involved in disulfidebonding, glycosylation sites, etc.).

Conformational epitopes present in the NS3/4a region are readilyidentified using methods discussed above. Moreover, the presence orabsence of a conformational epitope in a given polypeptide can bereadily determined through screening the antigen of interest with anantibody (polyclonal serum or monoclonal to the conformational epitope)and comparing its reactivity to that of a denatured version of theantigen which retains only linear epitopes (if any). In such screeningusing polyclonal antibodies, it may be advantageous to absorb thepolyclonal serum first with the denatured antigen and see if it retainsantibodies to the antigen of interest. Additionally, in the case ofNS3/4a, a molecule which preserves the native conformation will alsohave protease and, optionally, helicase enzymatic activities. Suchactivities can be detected using enzymatic assays, as described furtherbelow.

Preferably, a conformational epitope is produced recombinantly and isexpressed in a cell from which it is extractable under conditions whichpreserve its desired structural features, e.g. without denaturation ofthe epitope. Such cells include bacteria, yeast, insect, and mammaliancells. Expression and isolation of recombinant conformational epitopesfrom the HCV polyprotein are described in e.g., InternationalPublication Nos. WO 96/04301, WO 94/01778, WO 95/33053, WO 92/08734,which applications are herein incorporated by reference in theirentirety. Alternatively, it is possible to express the antigens andfurther renature the protein after recovery. It is also understood thatchemical synthesis may also provide conformational antigen mimitopesthat cross-react with the “native” antigen's conformational epitope.

The term “multiple epitope fusion antigen” or “MEFA” as used hereinintends a polypeptide in which multiple HCV antigens are part of asingle, continuous chain of amino acids, which chain does not occur innature. The HCV antigens may be connected directly to each other bypeptide bonds or may be separated by intervening amino acid sequences.The fusion antigens may also contain sequences exogenous to the HCVpolyprotein. Moreover, the HCV sequences present may be from multiplegenotypes and/or isolates of HCV. Examples of particular MEFAs for usein the present immunoassays are detailed in, e.g., InternationalPublication No. WO 97/44469, incorporated herein by reference in itsentirety, and are described further below.

An “antibody” intends a molecule that, through chemical or physicalmeans, specifically binds to a polypeptide of interest. Thus, forexample, an HCV core antibody is a molecule that specifically binds tothe HCV core protein. The term “antibody” as used herein includesantibodies obtained from both polyclonal and monoclonal preparations, aswell as, the following: hybrid (chimeric) antibody molecules (see, forexample, Winter et al. (1991) Nature 349:293-299; and U.S. Pat. No.4,816,567); F(ab′)₂ and F(ab) fragments; Fv molecules (non-covalentheterodimers, see, for example, Inbar et al. (1972) Proc Natl Acad SciUSA 69:2659-2662; and Ehrlich et al. (1980) Biochem 19:4091-4096);single-chain Fv molecules (sFv) (see, for example, Huston et al. (1988)Proc Natl Acad Sci USA 85:5879-5883); dimeric and trimeric antibodyfragment constructs; minibodies (see, e.g., Pack et al. (1992) Biochem31:1579-1584; Cumber et al. (1992) J Immunology 149B: 120-126);humanized antibody molecules (see, for example, Riechmann et al. (1988)Nature 332:323-327; Verhoeyan et al. (1988) Science 239:1534-1536; andU.K. Patent Publication No. GB 2,276,169, published Sep. 21, 1994); and,any functional fragments obtained from such molecules, wherein suchfragments retain immunological binding properties of the parent antibodymolecule.

As used herein, the term “monoclonal antibody” refers to an antibodycomposition having a homogeneous antibody population. The term is notlimited regarding the species or source of the antibody, nor is itintended to be limited by the manner in which it is made. Thus, the termencompasses antibodies obtained from murine hybridomas, as well as humanmonoclonal antibodies obtained using human rather than murinehybridomas. See, e.g., Cote, et al. Monclonal Antibodies and CancerTherapy, Alan R. Liss, 1985, p. 77.

By “isolated” is meant, when referring to a polypeptide, that theindicated molecule is separate and discrete from the whole organism withwhich the molecule is found in nature or is present in the substantialabsence of other biological macro-molecules of the same type. The term“isolated” with respect to a polynucleotide is a nucleic acid moleculedevoid, in whole or part, of sequences normally associated with it innature; or a sequence, as it exists in nature, but having heterologoussequences in association therewith; or a molecule disassociated from thechromosome.

By “equivalent antigenic determinant” is meant an antigenic determinantfrom different sub-species or strains of HCV, such as from strains 1, 2,or 3 of HCV. More specifically, epitopes are known, such as 5-1-1, andsuch epitopes vary between the strains 1, 2, and 3. Thus, the epitope5-1-1 from the three different strains are equivalent antigenicdeterminants and thus are “copies” even though their sequences are notidentical. In general the amino acid sequences of equivalent antigenicdeterminants will have a high degree of sequence homology, e.g., aminoacid sequence homology of more than 30%, preferably more than 40%, whenthe two sequences are aligned.

“Homology” refers to the percent similarity between two polynucleotideor two polypeptide moieties. Two DNA, or two polypeptide sequences are“substantially homologous” to each other when the sequences exhibit atleast about 50%, preferably at least about 75%, more preferably at leastabout 80%-85%, preferably at least about 90%, and most preferably atleast about 95%-98% sequence similarity over a defined length of themolecules. As used herein, substantially homologous also refers tosequences showing complete identity to the specified DNA or polypeptidesequence.

In general, “identity” refers to an exact nucleotide-to-nucleotide oramino acid-to-amino acid correspondence of two polynucleotides orpolypeptide sequences, respectively. Percent identity can be determinedby a direct comparison of the sequence information between two moleculesby aligning the sequences, counting the exact number of matches betweenthe two aligned sequences, dividing by the length of the shortersequence, and multiplying the result by 100.

Readily available computer programs can be used to aid in the analysisof homology and identity, such as ALIGN, Dayhoff, M. O. in Atlas ofProtein Sequence and Structure M. O. Dayhoff ed., 5 Suppl. 3:353-358,National biomedical Research Foundation, Washington, D.C., which adaptsthe local homology algorithm of Smith and Waterman Advances in Appl.Math. 2:482-489, 1981 for peptide analysis. Programs for determiningnucleotide sequence homology are available in the Wisconsin SequenceAnalysis Package, Version 8 (available from Genetics Computer Group,Madison, Wis.) for example, the BESTFIT, FASTA and GAP programs, whichalso rely on the Smith and Waterman algorithm. These programs arereadily utilized with the default parameters recommended by themanufacturer and described in the Wisconsin Sequence Analysis Packagereferred to above. For example, percent homology of a particularnucleotide sequence to a reference sequence can be determined using thehomology algorithm of Smith and Waterman with a default scoring tableand a gap penalty of six nucleotide positions.

Another method of establishing percent homology in the context of thepresent invention is to use the MPSRCH package of programs copyrightedby the University of Edinburgh, developed by John F. Collins and ShaneS. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View,Calif.). From this suite of packages the Smith-Waterman algorithm can beemployed where default parameters are used for the scoring table (forexample, gap open penalty of 12, gap extension penalty of one, and a gapof six). From the data generated the “Match” value reflects “sequencehomology.” Other suitable programs for calculating the percent identityor similarity between sequences are generally known in the art, forexample, another alignment program is BLAST, used with defaultparameters. For example, BLASTN and BLASTP can be used using thefollowing default parameters: genetic code=standard; filter=none;strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs can be found at thefollowing internet address: http://www.ncbi.nlm.gov/cgi-bin/BLAST.

Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions which form stable duplexes betweenhomologous regions, followed by digestion with single-stranded-specificnuclease(s), and size determination of the digested fragments. DNAsequences that are substantially homologous can be identified in aSouthern hybridization experiment under, for example, stringentconditions, as defined for that particular system. Defining appropriatehybridization conditions is within the skill of the art. See, e.g.,Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization,supra.

A “coding sequence” or a sequence which “encodes” a selectedpolypeptide, is a nucleic acid molecule which is transcribed (in thecase of DNA) and translated (in the case of mRNA) into a polypeptide invitro or in vivo when placed under the control of appropriate regulatorysequences. The boundaries of the coding sequence are determined by astart codon at the 5′ (amino) terminus and a translation stop codon atthe 3′ (carboxy) terminus. A transcription termination sequence may belocated 3′ to the coding sequence.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their desiredfunction. Thus, a given promoter operably linked to a coding sequence iscapable of effecting the expression of the coding sequence when theproper transcription factors, etc., are present. The promoter need notbe contiguous with the coding sequence, so long as it functions todirect the expression thereof. Thus, for example, interveninguntranslated yet transcribed sequences can be present between thepromoter sequence and the coding sequence, as can transcribed introns,and the promoter sequence can still be considered “operably linked” tothe coding sequence.

“Recombinant” as used herein to describe a nucleic acid molecule means apolynucleotide of genomic, cDNA, viral, semisynthetic, or syntheticorigin which, by virtue of its origin or manipulation is not associatedwith all or a portion of the polynucleotide with which it is associatedin nature. The term “recombinant” as used with respect to a protein orpolypeptide means a polypeptide produced by expression of a recombinantpolynucleotide. In general, the gene of interest is cloned and thenexpressed in transformed organisms, as described further below. The hostorganism expresses the foreign gene to produce the protein underexpression conditions.

A “control element” refers to a polynucleotide sequence which aids inthe expression of a coding sequence to which it is linked. The termincludes promoters, transcription termination sequences, upstreamregulatory domains, polyadenylation signals, untranslated regions,including 5′-UTRs and 3′-UTRs and when appropriate, leader sequences andenhancers, which collectively provide for the transcription andtranslation of a coding sequence in a host cell.

A “promoter” as used herein is a DNA regulatory region capable ofbinding RNA polymerase in a host cell and initiating transcription of adownstream (3′ direction) coding sequence operably linked thereto. Forpurposes of the present invention, a promoter sequence includes theminimum number of bases or elements necessary to initiate transcriptionof a gene of interest at levels detectable above background. Within thepromoter sequence is a transcription initiation site, as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase. Eucaryotic promoters will often, but not always, contain“TATA” boxes and “CAT” boxes.

A control sequence “directs the transcription” of a coding sequence in acell when RNA polymerase will bind the promoter sequence and transcribethe coding sequence into mRNA, which is then translated into thepolypeptide encoded by the coding sequence.

“Expression cassette” or “expression construct” refers to an assemblywhich is capable of directing the expression of the sequence(s) orgene(s) of interest. The expression cassette includes control elements,as described above, such as a promoter which is operably linked to (soas to direct transcription of) the sequence(s) or gene(s) of interest,and often includes a polyadenylation sequence as well. Within certainembodiments of the invention, the expression cassette described hereinmay be contained within a plasmid construct. In addition to thecomponents of the expression cassette, the plasmid construct may alsoinclude, one or more selectable markers, a signal which allows theplasmid construct to exist as single-stranded DNA (e.g., a M13 origin ofreplication), at least one multiple cloning site, and a “mammalian”origin of replication (e.g., a SV40 or adenovirus origin ofreplication).

“Transformation,” as used herein, refers to the insertion of anexogenous polynucleotide into a host cell, irrespective of the methodused for insertion: for example, transformation by direct uptake,transfection, infection, and the like. For particular methods oftransfection, see further below. The exogenous polynucleotide may bemaintained as a nonintegrated vector, for example, an episome, oralternatively, may be integrated into the host genome.

A “host cell” is a cell which has been transformed, or is capable oftransformation, by an exogenous DNA sequence.

As used herein, a “biological sample” refers to a sample of tissue orfluid isolated from a subject, that commonly includes antibodiesproduced by the subject. Typical samples that include such antibodiesare known in the art and include but not limited to, blood, plasma,serum, fecal matter, urine, bone marrow, bile, spinal fluid, lymphfluid, samples of the skin, secretions of the skin, respiratory,intestinal, and genitourinary tracts, tears, saliva, milk, blood cells,organs, biopsies and also samples of in vitro cell culture constituentsincluding but not limited to conditioned media resulting from the growthof cells and tissues in culture medium, e.g., recombinant cells, andcell components.

“Common solid support” intends a single solid matrix to which the HCVpolypeptides used in the subject immunoassays are bound covalently or bynoncovalent means such as hydrophobic adsorption.

“Immunologically reactive” means that the antigen in question will reactspecifically with anti-HCV antibodies present in a biological samplefrom an HCV-infected individual.

“Immune complex” intends the combination formed when an antibody bindsto an epitope on an antigen.

As used herein, the terms “label” and “detectable label” refer to amolecule capable of detection, including, but not limited to,radioactive isotopes, fluorescers, chemiluminescers, chromophores,enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors,chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin,avidin, strepavidin or haptens) and the like. The term “fluorescer”refers to a substance or a portion thereof which is capable ofexhibiting fluorescence in the detectable range. Particular examples oflabels which may be used under the invention include, but are notlimited to, horse radish peroxidase (HRP), fluorescein, FITC, rhodamine,dansyl, umbelliferone, dimethyl acridinium ester (DMAE), Texas red,luminol, NADPH and α-β-galactosidase.

II. Modes of Carrying Out the Invention

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular formulationsor process parameters as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting.

Although a number of compositions and methods similar or equivalent tothose described herein can be used in the practice of the presentinvention, the preferred materials and methods are described herein.

As noted above, the present invention is based on the discovery of noveldiagnostic methods for accurately detecting early HCV infection. Themethods rely on the identification and use of highly immunogenic HCVantigens which are present during the early stages of HCVseroconversion, thereby increasing detection accuracy and reducing theincidence of false results. In particular, the immunoassays describedherein utilize highly immunogenic conformational epitopes derived fromthe NS3/4a region of the HCV polyprotein, and multiple epitope fusionantigens comprising various HCV polypeptides, either from the same ordifferent HCV genotypes and isolates, such as multiple immunodominantepitopes, for example, major linear epitopes of HCV core, E1, E2, NS3,5-1-1, c100-3 and NS5 sequences. The methods can be convenientlypracticed in a single assay, using any of the several assay formatsdescribed below, such as but not limited to, assay formats which utilizea solid support to which the HCV antigens are bound.

The NS3/4a region of the HCV polyprotein has been described and theamino acid sequence and overall structure of the protein are disclosedin, e.g., Yao et al., Structure (November 1999) 7:1353-1363; Sali etal., Biochem. (1998) 37:3392-3401; and Bartenschlager, R., J. ViralHepat. (1999) 6:165-181. See, also, Dasmahapatra et al., U.S. Pat. No.5,843,752, incorporated herein by reference in its entirety. The subjectimmunoassays utilize at least one conformational epitope derived fromthe NS3/4a region that exists in the conformation as found in thenaturally occurring HCV particle or its infective product, as evidencedby the preservation of protease and, optionally, helicase enzymaticactivities normally displayed by the NS3/4a gene product and/orimmunoreactivity of the antigen with antibodies in a biological samplefrom an HCV-infected subject, and a loss of the epitope'simmunoreactivity upon denaturation of the antigen. For example, theconformational epitope can be disrupted by heating, changing the pH toextremely acid or basic, or by adding known organic denaturants, such asdithiothreitol (DTT) or an appropriate detergent. See, e.g., ProteinPurification Methods, a practical approach (E. L. V. Harris and S. Angaleds., IRL Press) and the denatured product compared to the product whichis not treated as above.

Protease and helicase activity may be determined using standard enzymeassays well known in the art. For example, protease activity may bedetermined using the procedure described below in the examples, as wellas using assays well known in the art. See, e.g., Takeshita et al.,Anal. Biochem. (1997) 247:242-246; Kakiuchi et al., J. Biochem. (1997)122:749-755; Sali et al., Biochemistry (1998) 37:3392-3401; Cho et al.,J. Virol. Meth. (1998) 72:109-115; Cerretani et al., Anal. Biochem.(1999) 266:192-197; Zhang et al., Anal. Biochem. (1999) 270:268-275;Kakiuchi et al., J. Virol. Meth. (1999) 80:77-84; Fowler et al., J.Biomol. Screen. (2000) 5:153-158; and Kim et al., Anal. Biochem. (2000)284:42-48. Similarly, helicase activity assays are well known in the artand helicase activity of an NS3/4a epitope may be determined using, forexample, an ELISA assay, as described in, e.g., Hsu et al., Biochem.Biophys. Res. Commun. (1998) 253:594-599; a scintillation proximityassay system, as described in Kyono et al., Anal. Biochem. (1998)257:120-126; high throughput screening assays as described in, e.g.,Hicham et al., Antiviral Res. (2000) 46:181-193 and Kwong et al.,Methods Mol. Med. (2000) 24:97-116; as well as by other assay methodsknown in the art. See, e.g., Khu et al., J. Virol. (2001) 75:205-214;Utama et al., Virology (2000) 273:316-324; Paolini et al., J. Gen.Virol. (2000) 81:1335-1345; Preugschat et al., Biochemistry (2000)39:5174-5183; Preugschat et al., Methods Mol. Med. (1998) 19:353-364;and Hesson et al., Biochemistry (2000) 39:2619-2625.

The length of the antigen is sufficient to maintain an immunoreactiveconformational epitope. Often, the polypeptide containing the antigenused will be almost full-length, however, the polypeptide may also betruncated to, for example, increase solubility or to improve secretion.Generally, the conformational epitope found in NS3/4a is expressed as arecombinant polypeptide in a cell and this polypeptide provides theepitope in a desired form, as described in detail below.

A representative amino acid sequence for the NS3/4a polypeptide is shownin FIGS. 3A through 3D. The amino acid sequence shown at positions 2-686of the figure corresponds to amino acid positions 1027-1711 of HCV-1. Aninitiator codon (ATG) coding for Met, is shown as position 1.Additionally, the Thr normally occurring at position 1428 of HCV-1(amino acid position 403 of FIG. 3) is mutated to Pro, and the Sernormally occurring at position 1429 of HCV-1 (amino acid position 404 ofFIG. 3) is mutated to Ile. However, either the native sequence, with orwithout an N-terminal Met, the depicted analog, with or without theN-terminal Met, or other analogs and fragments can be used in thesubject assays, so long as the epitope is produced using a method thatretains or reinstates its native conformation such that proteaseactivity, and optionally, helicase activity is retained. Dasmahapatra etal., U.S. Pat. No. 5,843,752 and Zhang et al., U.S. Pat. No. 5,990,276,both describe analogs of NS3/4a.

The NS3 protease of NS3/4a is found at about positions 1027-1207,numbered relative to HCV-1 (see, Choo et al., Proc. Natl. Acad. Sci. USA(1991) 88:2451-2455), positions 2-182 of FIG. 3. The structure of theNS3 protease and active site are known. See, e.g., De Francesco et al.,Antivir. Ther. (1998) 3:99-109; Koch et al., Biochemistry (2001)40:631-640. Changes to the native sequence that will normally betolerated will be those outside of the active site of the molecule.Particularly, it is desirable to maintain amino acids 1- or 2-155 ofFIG. 3, with little or only conservative substitutions. Amino acidsoccurring beyond 155 will tolerate greater changes. Additionally, iffragments of the NS3/4a sequence found in FIG. 3 are used, thesefragments will generally include at least amino acids 1- or 2-155,preferably amino acids 1- or 2-175, and most preferably amino acids 1-or 2-182, with or without the N-terminal Met. The helicase domain isfound at about positions 1193-1657 of HCV-1 (positions 207-632 of FIG.3). Thus, if helicase activity is desired, this portion of the moleculewill be maintained with little or only conservative changes. One ofskill in the art can readily determine other regions that will toleratechange based on the known structure of NS3/4a.

The immunoassays described herein also utilize multiple epitope fusionantigens (termed “MEFAs”), as described in International Publication No.WO 97/44469. Such MEFAs include multiple epitopes derived from two ormore of the various viral regions of the HCV polyprotein shown in FIG. 1and Table 1. In particular, as shown in FIG. 1 and Table 1, An HCVpolyprotein, upon cleavage, produces at least ten distinct products, inthe order of— Core-E1-E2-p7-NS2-NS3-NS4a-NS4b-NS5a-NS5b-COOH. The corepolypeptide occurs at positions 1-191, numbered relative to HCV-1 (see,Choo et al. (1991) Proc. Natl. Acad. Sci. USA 88:2451-2455, for theHCV-1 genome). This polypeptide is further processed to produce an HCVpolypeptide with approximately amino acids 1-173. The envelopepolypeptides, E1 and E2, occur at about positions 192-383 and 384-746,respectively. The P7 domain is found at about positions 747-809. NS2 isan integral membrane protein with proteolytic activity and is found atabout positions 810-1026 of the polyprotein. NS2, either alone or incombination with NS3 (found at about positions 1027-1657), cleaves theNS2-NS3 sissle bond which in turn generates the NS3 N-terminus andreleases a large polyprotein that includes both serine protease and RNAhelicase activities. The NS3 protease, found at about positions1027-1207, serves to process the remaining polyprotein. The helicaseactivity is found at about positions 1193-1657. Completion ofpolyprotein maturation is initiated by autocatalytic cleavage at theNS3-NS4a junction, catalyzed by the NS3 serine protease. SubsequentNS3-mediated cleavages of the HCV polyprotein appear to involverecognition of polyprotein cleavage junctions by an NS3 molecule ofanother polypeptide. In these reactions, NS3 liberates an NS3 cofactor(NS4a, found about positions 1658-1711), two proteins (NS4b found atabout positions 1712-1972, and NS5a found at about positions 1973-2420),and an RNA-dependent RNA polymerase (NS5b found at about positions2421-3011).

TABLE 1 Domain Approximate Boundaries* C (core)  1-191 E1 192-383 E2384-746 P7 747-809 NS2  810-1026 NS3 1027-1657 NS4a 1658-1711 NS4b1712-1972 NS5a 1973-2420 NS5b 2421-3011 *Numbered relative to HCV-1.See, Choo et al. (1991) Proc. Natl. Acad. Sci. USA 88: 2451-2455.

The multiple HCV antigens are part of a single, continuous chain ofamino acids, which chain does not occur in nature. Thus, the linearorder of the epitopes is different than their linear order in the genomein which they occur. The linear order of the sequences of the MEFAs foruse herein is preferably arranged for optimum antigenicity. Preferably,the epitopes are from more than one HCV strain, thus providing the addedability to detect multiple strains of HCV in a single assay. Thus, theMEFAs for use herein may comprise various immunogenic regions derivedfrom the polyprotein described above. Moreover, a protein resulting froma frameshift in the core region of the polyprotein, such as described inInternational Publication No. WO 99/63941, may be used in the MEFAs. Ifdesired, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more of one or moreepitopes derived from the HCV polyprotein may occur in the fusionprotein.

For example, epitopes derived from, e.g., the hypervariable region ofE2, such as a region spanning amino acids 384-410 or 390-410, can beincluded in the MEFA antigen. A particularly effective E2 epitope is onewhich includes a consensus sequence derived from this region, such asthe consensus sequenceGly-Ser-Ala-Ala-Arg-Thr-Thr-Ser-Gly-Phe-Val-Ser-Leu-Phe-Ala-Pro-Gly-Ala-Lys-Gln-Asn(SEQ ID NO:5), which represents a consensus sequence for amino acids390-410 of the HCV type 1 genome. A representative E2 epitope present ina MEFA of the invention can comprise a hybrid epitope spanning aminoacids 390-444. Such a hybrid E2 epitope can include a consensus sequencerepresenting amino acids 390-410 fused to the native amino acid sequencefor amino acids 411-444 of HCV E2.

Additionally, the antigens may be derived from various HCV strains.Multiple viral strains of HCV are known, and epitopes derived from anyof these strains can be used in a fusion protein. It is well known thatany given species of organism varies from one individual organism toanother and further that a given organism such as a virus can have anumber of different strains. For example, as explained above, HCVincludes at least 6 genotypes. Each of these genotypes includesequivalent antigenic determinants. More specifically, each strainincludes a number of antigenic determinants that are present on allstrains of the virus but are slightly different from one viral strain toanother. For example, HCV includes the antigenic determinant known as5-1-1 (See, FIG. 1). This particular antigenic determinant appears inthree different forms on the three different viral strains of HCV.Accordingly, in a preferred embodiment of the invention all three formsof 5-1-1 appear on the multiple epitope fusion antigen used in thesubject immunoassays. Similarly, equivalent antigenic determinants fromthe core region of different HCV strains may also be present. Ingeneral, equivalent antigenic determinants have a high degree ofhomology in terms of amino acid sequence which degree of homology isgenerally 30% or more, preferably 40% or more, when aligned. Themultiple copy epitope of the present invention can also include multiplecopies which are exact copies of the same epitope.

FIGS. 4 and 6A-6C show representative MEFAs for use in the presentinvention which are derived from HCV. However, it is to be understoodthat other epitopes derived from the HCV genome will also find use withthe present assays.

The DNA sequence and corresponding amino acid sequence of arepresentative multiple epitope fusion antigen, MEFA 7.1, is shown inFIGS. 5A through 5F. The general structural formula for MEFA 7.1 isshown in FIG. 4 and is as follows: hSOD-E1(type 1)-E2 HVR consensus(type1a)-E2 HVR consensus(types 1 and 2)-helicase(type 1)-5-1-1(type1)-5-1-1(type 3)-5-1-1(type 2)-c100(type 1)-NS5(type1)-NS5(type1)-core(types 1+2)-core(types 1+2). This multiple copyepitope includes the following amino acid sequence, numbered relative toHCV-1 (the numbering of the amino acids set forth below follows thenumbering designation provided in Choo, et al. (1991) Proc. Natl. Acad.Sci. USA 88:2451-2455, in which amino acid #1 is the first methionineencoded by the coding sequence of the core region): amino acids 1-156 ofsuperoxide dismutase (SOD, used to enhance recombinant expression of theprotein); amino acids 303 to 320 of the polyprotein from the E1 region;amino acids 390 to 410 of the polyprotein, representing a consensussequence for the hypervariable region of HCV-1 a E2; amino acids 384 to414 of the polyprotein from region E2, representing a consensus sequencefor the E2 hypervariable regions of HCV-1 and HCV-2; amino acids1193-1658 of the HCV-1 polyprotein which define the helicase; threecopies of an epitope from 5-1-1, amino acids 1689-1735, one from HCV-1,one from HCV-3 and one from HCV-2, which copies are equivalent antigenicdeterminants from the three different viral strains of HCV; HCVpolypeptide C100 of HCV-1, amino acids 1901-1936 of the polyprotein; twoexact copies of an epitope from the NS5 region of HCV-1, each with aminoacids 2278 to 2313 of the HCV polyprotein; and two copies of an epitopefrom the core region, one from HCV-1 and one from HCV-2, which copiesare equivalent antigenic determinants represented by amino acids 9 to32, 39-42 and 64-88 of HCV-1 and 67-84 of HCV-2.

Table 2 shows the amino acid positions of the various epitopes withreference to FIGS. 5A through 5F herein.

TABLE 2 MEFA 7.1 mefa aa# 5′ end site epitope hcv aa# strain  1-156 Nco1hSOD 159-176 EcoR1 E1 303-320 1 179-199 Hind111 E2 HVR1a 390-410 1consensus 200-230 E2 HVR1 + 2 384-414 1 + 2 consensus 231-696 Sal1Helicase 1193-1658 1 699-745 Sph1 5-1-1 1689-1735 1 748-794 Nru1 5-1-11689-1735 3 797-843 Cla1 5-1-1 1689-1735 2 846-881 Ava1 C100 1901-1936 1884-919 Xba1 NS5 2278-2313 1 922-957 Bgl11 NS5 2278-2313 1  958-1028Nco1 core 9-32, 39-42 1 epitopes 64-88 1 67-84 2 1029-1099 Bal1 core9-32, 39-42, 1 epitopes 64-88 1 67-84 2

In one embodiment of the invention, depicted in FIG. 2, a rapid captureligand immunoassay is performed using a conformational epitope fromNS3/4a and one or more multiple epitope fusion antigens, such as MEFA7.1. The sample is combined with the antigens, which may be present on asolid support, as described further below. If the sample is infectedwith HCV, HCV antibodies to those epitopes present on the solid supportwill bind to the solid support components. Detection is by theattachment of a detectable marker (such as horse radish peroxidase (HRP)as shown in FIG. 2) to a member of the antigen/antibody complex.Attachment may be by covalent means or by subsequent binding ofdetectably labeled antibodies, such as in a standard sandwich assay, orby enzyme reaction, the product of which reaction is detectable. Thedetectable marker may include, but is not limited to, a chromophore, anantibody, an antigen, an enzyme, an enzyme reactive compound whosecleavage product is detectable, rhodamine or rhodamine derivative,biotin, avidin, strepavidin, a fluorescent compound, a chemiluminescentcompound, such as dimethyl acridinium ester (DMAE, Ciba CorningDiagnostics Corp.), derivatives and/or combinations of these markers. Adetectably labeled anti-human antibody, capable of detecting a human IgGmolecule present, can be conveniently used.

In order to further an understanding of the invention, a more detaileddiscussion is provided below regarding production of polypeptides foruse in the immunoassays and methods of conducting the immunoassays.

Production of Antigens for Use in the HCV Immunoassays

As explained above, the molecules of the present invention are generallyproduced recombinantly. Thus, polynucleotides encoding HCV antigens foruse with the present invention can be made using standard techniques ofmolecular biology. For example, polynucleotide sequences coding for theabove-described molecules can be obtained using recombinant methods,such as by screening cDNA and genomic libraries from cells expressingthe gene, or by deriving the gene from a vector known to include thesame. Furthermore, the desired gene can be isolated directly from viralnucleic acid molecules, using techniques described in the art, such asin Houghton et al., U.S. Pat. No. 5,350,671. The gene of interest canalso be produced synthetically, rather than cloned. The molecules can bedesigned with appropriate codons for the particular sequence. Thecomplete sequence is then assembled from overlapping oligonucleotidesprepared by standard methods and assembled into a complete codingsequence. See, e.g., Edge (1981) Nature 292:756; Nambair et al. (1984)Science 223:1299; and Jay et al. (1984) J. Biol. Chem. 259:6311.

Thus, particular nucleotide sequences can be obtained from vectorsharboring the desired sequences or synthesized completely or in partusing various oligonucleotide synthesis techniques known in the art,such as site-directed mutagenesis and polymerase chain reaction (PCR)techniques where appropriate. See, e.g., Sambrook, supra. In particular,one method of obtaining nucleotide sequences encoding the desiredsequences is by annealing complementary sets of overlapping syntheticoligonucleotides produced in a conventional, automated polynucleotidesynthesizer, followed by ligation with an appropriate DNA ligase andamplification of the ligated nucleotide sequence via PVR. See, e.g.,Jayaraman et al. (1991) Proc. Natl. Acad. Sci. USA 88:4084-4088.Additionally, oligonucleotide directed synthesis (Jones et al. (1986)Nature 54:75-82), oligonucleotide directed mutagenesis of pre-existingnucleotide regions (Riechmann et al. (1988) Nature 332:323-327 andVerhoeyen et al. (1988) Science 239:1534-1536), and enzymatic filling-inof gapped oligonucleotides using T₄ DNA polymerase (Queen et al. (1989)Proc. Natl. Acad. Sci. USA 86:10029-10033) can be used under theinvention to provide molecules having altered or enhancedantigen-binding capabilities, and/or reduced immunogenicity.

Once coding sequences have been prepared or isolated, such sequences canbe cloned into any suitable vector or replicon. Numerous cloning vectorsare known to those of skill in the art, and the selection of anappropriate cloning vector is a matter of choice. Suitable vectorsinclude, but are not limited to, plasmids, phages, transposons, cosmids,chromosomes or viruses which are capable of replication when associatedwith the proper control elements.

The coding sequence is then placed under the control of suitable controlelements, depending on the system to be used for expression. Thus, thecoding sequence can be placed under the control of a promoter, ribosomebinding site (for bacterial expression) and, optionally, an operator, sothat the DNA sequence of interest is transcribed into RNA by a suitabletransformant. The coding sequence may or may not contain a signalpeptide or leader sequence which can later be removed by the host inpost-translational processing. See, e.g., U.S. Pat. Nos. 4,431,739;4,425,437; 4,338,397.

In addition to control sequences, it may be desirable to add regulatorysequences which allow for regulation of the expression of the sequencesrelative to the growth of the host cell. Regulatory sequences are knownto those of skill in the art, and examples include those which cause theexpression of a gene to be turned on or off in response to a chemical orphysical stimulus, including the presence of a regulatory compound.Other types of regulatory elements may also be present in the vector.For example, enhancer elements may be used herein to increase expressionlevels of the constructs. Examples include the SV40 early gene enhancer(Dijkema et al. (1985) EMBO J. 4:761), the enhancer/promoter derivedfrom the long terminal repeat (LTR) of the Rous Sarcoma Virus (Gorman etal. (1982) Proc. Natl. Acad. Sci. USA 79:6777) and elements derived fromhuman CMV (Boshart et al. (1985) Cell 41:521), such as elements includedin the CMV intron A sequence (U.S. Pat. No. 5,688,688). The expressioncassette may further include an origin of replication for autonomousreplication in a suitable host cell, one or more selectable markers, oneor more restriction sites, a potential for high copy number and a strongpromoter.

An expression vector is constructed so that the particular codingsequence is located in the vector with the appropriate regulatorysequences, the positioning and orientation of the coding sequence withrespect to the control sequences being such that the coding sequence istranscribed under the “control” of the control sequences (i.e., RNApolymerase which binds to the DNA molecule at the control sequencestranscribes the coding sequence). Modification of the sequences encodingthe molecule of interest may be desirable to achieve this end. Forexample, in some cases it may be necessary to modify the sequence sothat it can be attached to the control sequences in the appropriateorientation; i.e., to maintain the reading frame. The control sequencesand other regulatory sequences may be ligated to the coding sequenceprior to insertion into a vector. Alternatively, the coding sequence canbe cloned directly into an expression vector which already contains thecontrol sequences and an appropriate restriction site.

As explained above, it may also be desirable to produce mutants oranalogs of the antigen of interest. This is particularly true withNS3/4a. Methods for doing so are described in, e.g., Dasmahapatra etal., U.S. Pat. No. 5,843,752 and Zhang et al., U.S. Pat. No. 5,990,276.Mutants or analogs of this and other HCV proteins for use in the subjectassays may be prepared by the deletion of a portion of the sequenceencoding the polypeptide of interest, by insertion of a sequence, and/orby substitution of one or more nucleotides within the sequence.Techniques for modifying nucleotide sequences, such as site-directedmutagenesis, and the like, are well known to those skilled in the art.See, e.g., Sambrook et al., supra; Kunkel, T. A. (1985) Proc. Natl.Acad. Sci. USA (1985) 82:448; Geisselsoder et al. (1987) BioTechniques5:786; Zoller and Smith (1983) Methods Enzymol. 100:468;Dalbie-McFarland et al. (1982) Proc. Natl. Acad. Sci USA 79:6409.

The molecules can be expressed in a wide variety of systems, includinginsect, mammalian, bacterial, viral and yeast expression systems, allwell known in the art.

For example, insect cell expression systems, such as baculovirussystems, are known to those of skill in the art and described in, e.g.,Summers and Smith, Texas Agricultural Experiment Station Bulletin No.1555 (1987). Materials and methods for baculovirus/insect cellexpression systems are commercially available in kit form from, interalia, Invitrogen, San Diego Calif. (“MaxBac” kit). Similarly, bacterialand mammalian cell expression systems are well known in the art anddescribed in, e.g., Sambrook et al., supra. Yeast expression systems arealso known in the art and described in, e.g., Yeast Genetic Engineering(Barr et al., eds., 1989) Butterworths, London.

A number of appropriate host cells for use with the above systems arealso known. For example, mammalian cell lines are known in the art andinclude immortalized cell lines available from the American Type CultureCollection (ATCC), such as, but not limited to, Chinese hamster ovary(CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidneycells (COS), human embryonic kidney cells, human hepatocellularcarcinoma cells (e.g., Hep G2), Madin-Darby bovine kidney (“MDBK”)cells, as well as others. Similarly, bacterial hosts such as E. coli,Bacillus subtilis, and Streptococcus spp., will find use with thepresent expression constructs. Yeast hosts useful in the presentinvention include inter alia, Saccharomyces cerevisiae, Candidaalbicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis,Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris,Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for usewith baculovirus expression vectors include, inter alia, Aedes aegypti,Autographa californica, Bombyx mori, Drosophila melanogaster, Spodopterafrugiperda, and Trichoplusia ni.

Nucleic acid molecules comprising nucleotide sequences of interest canbe stably integrated into a host cell genome or maintained on a stableepisomal element in a suitable host cell using various gene deliverytechniques well known in the art. See, e.g., U.S. Pat. No. 5,399,346.

Depending on the expression system and host selected, the molecules areproduced by growing host cells transformed by an expression vectordescribed above under conditions whereby the protein is expressed. Theexpressed protein is then isolated from the host cells and purified. Ifthe expression system secretes the protein into growth media, theproduct can be purified directly from the media. If it is not secreted,it can be isolated from cell lysates. The selection of the appropriategrowth conditions and recovery methods are within the skill of the art.

The production of various HCV antigens, including antigens used in themultiple epitope fusion proteins described above, has been described.See, e.g., Houghton et al., U.S. Pat. Nos. 5,350,671 and 5,683,864;Chien et al., J. Gastroent. Hepatol. (1993) 8:S33-39; Chien et al.,International Publication No. WO 93/00365; Chien, D. Y., InternationalPublication No. WO 94/01778; Chien et al., Proc. Natl. Acad. Sci. USA(1992) 89:10011-10015; Chien, D. Y., International Publication No. WO94/01778; and commonly owned, allowed U.S. patent application Ser. Nos.08/403,590 and 08/444,818, the disclosures of which are incorporatedherein by reference in their entireties.

Immunodiagnostic Assays

Once produced, the HCV antigens may be used in virtually any assayformat that employs a known antigen to detect antibodies. A commonfeature of all of these assays is that the antigen is contacted with thebody component suspected of containing HCV antibodies under conditionsthat permit the antigen to bind to any such antibodies present in thecomponent. Such conditions will typically be physiologic temperature, pHand ionic strength using an excess of antigen. The incubation of theantigen with the specimen is followed by detection of immune complexescomprised of the antigen.

Design of the immunoassays is subject to a great deal of variation, andmany formats are known in the art. Protocols may, for example, use solidsupports, or immunoprecipitation. Most assays involve the use of labeledantibody or polypeptide; the labels may be, for example, enzymatic,fluorescent, chemiluminescent, radioactive, or dye molecules, asdiscussed in detail above. Assays which amplify the signals from theimmune complex are also known; examples of which are assays whichutilize biotin and avidin, and enzyme-labeled and mediated immunoassays,such as ELISA assays.

The immunoassay may be, without limitation, a heterogenous or ahomogeneous format, and of a standard or competitive type. In aheterogeneous format, the polypeptide is typically bound to a solidmatrix or support to facilitate separation of the sample from thepolypeptide after incubation. A solid support, for the purposes of thisinvention, can be any material that is an insoluble matrix and can havea rigid or semi-rigid surface. Exemplary solid supports include, but arenot limited to, substrates such as nitrocellulose (e.g., in membrane ormicrotiter well form); polyvinylchloride (e.g., sheets or microtiterwells); polystyrene latex (e.g., beads or microtiter plates);polyvinylidine fluoride; diazotized paper; nylon membranes; activatedbeads, magnetically responsive beads, and the like. Particular supportsinclude plates, pellets, disks, capillaries, hollow fibers, needles,pins, solid fibers, cellulose beads, pore-glass beads, silica gels,polystyrene beads optionally cross-linked with divinylbenzene, graftedco-poly beads, polyacrylamide beads, latex beads, dimethylacrylamidebeads optionally crosslinked with N-N′-bis-acryloylethylenediamine, andglass particles coated with a hydrophobic polymer.

If desired, the molecules to be added to the solid support can readilybe functionalized to create styrene or acrylate moieties, thus enablingthe incorporation of the molecules into polystyrene, polyacrylate orother polymers such as polyimide, polyacrylamide, polyethylene,polyvinyl, polydiacetylene, polyphenylene-vinylene, polypeptide,polysaccharide, polysulfone, polypyrrole, polyimidazole, polythiophene,polyether, epoxies, silica glass, silica gel, siloxane, polyphosphate,hydrogel, agarose, cellulose, and the like.

In one context, a solid support is first reacted with the HCV antigens(collectively called “the solid-phase components” herein), undersuitable binding conditions such that the molecules are sufficientlyimmobilized to the support. Sometimes, immobilization to the support canbe enhanced by first coupling the antigen and/or antibody to a proteinwith better solid phase-binding properties. Suitable coupling proteinsinclude, but are not limited to, macromolecules such as serum albuminsincluding bovine serum albumin (BSA), keyhole limpet hemocyanin,immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteinswell known to those skilled in the art. Other reagents that can be usedto bind molecules to the support include polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers,and the like. Such molecules and methods of coupling these molecules toantigens, are well known to those of ordinary skill in the art. See,e.g., Brinkley, M. A. (1992) Bioconjugate Chem. 3:2-13; Hashida et al.(1984) J. Appl. Biochem. 6:56-63; and Anjaneyulu and Staros (1987)International J. of Peptide and Protein Res. 30:117-124.

After reacting the solid support with the solid-phase components, anynonimmobilized solid-phase components are removed from the support bywashing, and the support-bound components are then contacted with abiological sample suspected of containing HCV antibodies (collectivelycalled “ligand molecules” herein) under suitable binding conditions. IfHCV antibodies are present in the sample, they will form a complex withthe HCV antigens. After washing to remove any nonbound ligand molecules,detectably labeled anti-xenogenic (e.g., anti-human) antibodies, whichrecognize an epitope on anti-HCV antibodies, is added. These antibodiesbind due to complex formation.

In a homogeneous format, the test sample is incubated with thecombination of antigens in solution. For example, it may be underconditions that will precipitate any antigen-antibody complexes whichare formed. Both standard and competitive formats for homogeneous assaysare also known in the art.

In a standard format, the amount of HCV antibodies forming theantibody-antigen complex is directly monitored. This may be accomplishedby determining whether labeled anti-xenogenic (e.g., anti-human)antibodies which recognize an epitope on anti-HCV antibodies will binddue to complex formation. In a competitive format, the amount of HCVantibodies in the sample is deduced by monitoring the competitive effecton the binding of a known amount of labeled antibody (or other competingligand) in the complex.

More particularly, complexes formed comprising anti-HCV antibody (or, inthe case of competitive assays, the amount of competing antibody) aredetected by any of a number of known techniques, depending on theformat. For example, unlabeled HCV antibodies in the complex may bedetected using a conjugate of antixenogeneic Ig complexed with a label,(e.g., an enzyme label). In an immunoprecipitation or agglutinationassay format, the reaction between the HCV antigens and the antibodyforms a network that precipitates from the solution or suspension andforms a visible layer or film of precipitate. If no anti-HCV antibody ispresent in the test specimen, no visible precipitate is formed.

The above-described assay reagents, including the immunoassay solidsupport with bound antibodies and antigens, as well as antibodies andantigens to be reacted with the captured sample, can be provided inkits, with suitable instructions and other necessary reagents, in orderto conduct immunoassays as described above. The kit will normallycontain in separate containers the combination of antigens (eitheralready bound to a solid matrix or separate with reagents for bindingthem to the matrix), control antibody formulations (positive and/ornegative), labeled antibody when the assay format requires same andsignal generating reagents (e.g., enzyme substrate) if the label doesnot generate a signal directly. Instructions (e.g., written, tape, VCR,CD-ROM, etc.) for carrying out the assay usually will be included in thekit. The kit can also contain, depending on the particular immunoassayused, other packaged reagents and materials (i.e. wash buffers and thelike). Standard immunoassays, such as those described above, can beconducted using these kits.

III. Experimental

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

EXAMPLE 1 Construction of MEFA 7 and MEFA 7.1

The following example illustrates the preparation of a polyproteincassette of multiple HCV epitopes. The polyprotein expressed from themultiple epitope cassette is referred to herein as a Multiple EpitopeFusion Antigen (MEFA). Preferably, where an epitope is repeated, theextra copy or copies are tandemly arrayed in the same orientation. It isunderstood that the region of a viral coding sequence used as an epitopemay be varied slightly and still retain antigenic activity, and that theamino acid numbering designation may vary from strain to strain. Thus,the repeated epitopes may vary one from another in amino acid sequencedue to strain sequence variations and/or numbering designation.Preferably, the amino acid sequences of repeated epitopes within a MEFAare at least 30% homologous at the amino acid level, more preferably atleast 40% homologous at the amino acid level.

Unique restriction enzyme sites were introduced in order to connect theepitopes in the prescribed order and enhance the usefulness of theinvention by facilitating modifications in design of a chimeric antigen.The choice of restriction enzyme sites and cloning procedures arereadily determined by one of ordinary skill in the art of recombinantDNA technology. Preferably, the epitope junctions (amino acid sequencescreated between epitopes due to cloning) do not generate non-specificepitopes. Non-specific epitopes are, for example, non-HCV sequenceswhich do not exist adjacent to the HCV epitopes in nature. Non-specificepitopes may bind antibodies in a test sample causing false positiveassay results. Preferably, the multiple epitope fusion protein is testedfor false positive results due to such sequences generated at theepitope junctions. To avoid non-specific interactions with the MEFA dueto junction sequences, the DNA sequence encoding the junction may, forexample, be mutated such that non-specific interactions with the mutantamino acid sequence are reduced, and cloning of the epitope fragments ispossible.

The HCV MEFA 7 and 7.1 expression cassettes were constructed by cloningthe coding nucleotide sequences containing major epitopes in a tandemarray as shown in FIG. 4. A major epitope was chosen based on antibodyreaction frequency and reaction intensity (titer) to the epitope (Chein,D. Y. et al. (1994) Viral Hepatitis and Liver Disease, pp. 320-324). Thevarious DNA segments coding for the HCV epitopes were constructed by PCRamplification or by synthetic oligonucleotides. The amino acids in eachsegment are set forth in Table 2 above and shown in FIGS. 5A-5F. Thecomplete HCV-1 amino acid sequence (3011 amino acids) was determined byChoo, et al. (1991) Proc. Natl. Acad. Sci. USA 88:2451-2455, hereinincorporated by reference in its entirety. Oligonucleotides capable ofbinding to HCV are described in U.S. Pat. No. 5,350,671, hereinincorporated by reference in its entirety. The numbering of the aminoacids in epitopes of the invention follows the numbering designationprovided in Choo, et al., supra, in which amino acid number 1 is thefirst methionine encoded by the coding sequence of the core region,unless otherwise specified. For example, one epitope segment from NS5 isrepresented by amino acids 2278 to 2313 of the HCV polyprotein. Anepitope from the E1 region is represented by amino acids 303 to 320,numbered relative to the HCV-1 polyprotein.

MEFA 7 and 7.1 each contain epitopes from HCV-1, HCV-2 and HCV-3,allowing for detection of multiple types of a virus in a single assay.Methods of determining HCV serotype are found in WO 96/27153, hereinincorporated by reference in its entirety. For example, epitopes fromthe 5-1-1 region have been found to vary between serotypes of HCV. Acopy of each of the HCV type-specific 5-1-1 epitopes present in theMEFAs described herein allows binding of any of the HCV types that maybe present in the test biological sample.

The MEFA 7 and 7.1 constructs were genetically engineered for expressionin Saccharomyces cerevisiae, utilizing the yeast expression vectorpBS24.1 which contains 2μ sequences for autonomous replication in yeastand the yeast genes leu2-d and URA3 as selectable markers. Theβ-lactamase gene and the ColE1 origin of replication, required forplasmid replication in bacteria, were also present in this expressionvector. The yeast expression vector for MEFA 7, ps.MEFA7, wasconstructed first. Subsequently, the plasmid was modified in the codingregion for the HCV core epitopes to create the plasmid ps.MEFA7.1,encoding the MEFA 7.1 antigen.

In particular, as shown in FIGS. 7A through 7D, a yeast expressionplasmid for MEFA 7 was constructed as follows. First, a BamHI/HindIIIfragment of 1896 bp, encoding the ADH2/GAPDH hybrid promoter, hSOD(amino acids 1-156), followed by an E1 epitope (amino acids 303-320,HCV1 strain), was isolated from ps.MEFA6, the expression plasmidencoding MEFA 6, described in International Publication No. WO 97/44469.Next, a HindIIISphI synthetic DNA fragment of 269 bp which contains thecoding sequence for E2 HVR1a consensus epitope (amino acids 390-410,HCV-1), E2 HVR1+2 consensus epitope (amino acids 384-414, HCV1+2) andthe 5′ end of the helicase domain (amino acids 1193-1229, HCV-1) wascreated. An SphI/EclXI fragment of 1264 bp, encoding the remainder ofthe helicase domain (amino acids 1230-1651, HCV-1), was gel-purifiedfrom pTac5/HelI plasmid DNA. The HindIII/SphI synthetic DNA fragment andthe SphI/EclXI 1264 bp fragment were ligated into vectorpSP72new.HindIII/EclXI vector, to producepSP72new.HindIII/EclXI/e2.helicase. This vector was derived from pSP72an E. coli vector commercially available from Promega, Madison, Wis.(see, GenBank/EMBL Accession Number X65332). In particular, tofacilitate the subcloning of several MEFA 7 epitopes, a new multiplecloning site (MCS) polylinker was introduced, via synthetic oligos,between the SphI and BglII sites of pSP72. This new plasmid, namedpSP72new, was digested with HindIII and EclXI (also known as EagI),which have unique sites in the MCS. It was then dephosphorylated andgel-purified.

E. coli HB101 competent cells were transformed with the plasmid, andplated on Luria agar plates containing 100 μg/ml ampicillin. Desiredclones were identified using miniprep DNA analysis. After sequenceverification, the plasmid pSP72new.HindIII/EclXI/e2.helicase subclone #4was digested with HindIII and EclXI(EagI) to generate a 1534 bpfragment. The HindIII/EclXI fragment was gel-purified and ligated withEclXI/SphI oligonucleotides, encoding the last amino acids of thehelicase domain (amino acids 1651-1658, HCV-1), into a pGEM7HindIII/SphI vector. HB101 competent cells were transformed and platedon Luria-ampicillin (100 μg/ml). After identification of the desiredclones and sequence confirmation, pGEM7HindIII/SphI subclone #9 wasdigested with HindIII and SphI to generate a 1560 bp fragment, which wasgel purified (see, FIG. 7A).

To assemble the 3′ end portion of MEFA 7, the following steps wereperformed. The 5-1-1 epitopes (amino acids 1689-1735) from HCV-1, HCV-3and HCV-2 (in this order) were gel-isolated from ps.MEFA6, theexpression plasmid encoding MEFA 6, described in InternationalPublication No. WO 97/44469, as an SphI/AvaI fragment of 441 bp. Thisfragment was ligated with synthetic AvaI/XbaI oligonucleotides encodingthe c100 epitope (amino acids 1901-1936) into a pSP72new.SphI/XbaIvector. After HB101 transformation, clone identification, and sequenceverification, pSP72newSXi subclone #6 was digested with XbaI and NotI toprepare a pSP72newXbaI/NotI vector. Additionally, an XbaI/NcoI fragmentof 221 bp, which encoded a double repeat of an NS5 epitope (amino acids2278-2313, HCV-1), was isolated from ps.MEFA6. The Xba/NcoI fragment wasligated with NcoI/NotI oligonucleotides, encoding the first amino acidsof the HCV-1 core epitope, amino acids 9-17, in which the Lys atposition 9 was changed to Arg, and the Asn at position 11 was changed toThr, into the pSP72new XbaI/NotI vector prepared above. HB101transformants were analyzed and their plasmid DNA sequenced. A subclone,termed pSP72newSX/XNi #3, was digested with NotI/SalI to prepare avector for subsequent subcloning (see, FIG. 7B).

To complete the assembly of the 3′ end of MEFA 7, a double repeat of thesequence encoding a core epitope with amino acids 9-53 from HCV-1, plustwo genotype-specific epitopes of the core region (amino acids 64-88,HCV-1 and amino acids 67-84, HCV-2) were subcloned as follows intoNotI-SalI digested pSP72newSX/XNi subclone #3. First, a NotI/XmnIfragment of 92 bp encoding amino acids 18-51 of a core epitope wasisolated from pd.Core191RT clone #20. Plasmid pd.Core191RT wasconstructed by ligating into the pBS24.1 BamHI-SalI yeast expressionvector, a 1365 bp BamHI-NcoI fragment for the ADH2/GAPDH promoter, and a615 bp NcoI-SalI fragment encoding the first 191 amino acids of HCV-1core with amino acid 9 mutated from Lys to Arg and amino acid 11 mutatedfrom Asn to Thr. The 615 bp NcoI-SalI fragment was derived from an E.coli expression vector in which the core sequence for amino acids 1-191,with the same two mutations described above, had been cloned.

The 92 bp NotI/XmnI fragment was ligated with a pSP72newNot/Kpn vectorand with XmnI/KpnI oligonucleotides which encode the 3′ end of thecomplete core epitope. After sequence verification of the positiveclones, pSP72newNKi subclone #4 was digested with NotI and KpnI, and a224 bp fragment was gel-isolated. This NotI/KpnI fragment was ligatedwith 284 bp of oligonucleotides (KpnI-SalI ends) encoding a completerepeat of the core epitope described above into the pSP72newSX/XNiNotI/SalI vector described above. After HB101 transformation, cloneidentification and sequence verification, pSP72newSX/XN/NSi subclone #18was digested with SphI and SalI and a fragment of 1317 bp wasgel-isolated (see, FIG. 7C).

Lastly, the following fragments, described above, were ligated into thepBS24.1 BamHI/SalI yeast expression vector to create ps.MEFA7 (see, FIG.7D):

the BamHI/HindIII fragment of 1896 bp (FIG. 7A)

the HindIII/SphI fragment of 1560 bp (FIG. 7A)

the SphI/SalI fragment of 1317 bp (FIG. 7C)

S. cerevisiae strain AD3 was transformed with ps.MEFA7 and singletransformants were checked for expression after depletion of glucose inthe medium. The recombinant protein was expressed at high levels inyeast, as detected by Coomassie blue staining. In particular, yeastcells were transformed with the MEFA expression plasmid using a lithiumacetate protocol. Ura transformants were streaked for single coloniesand patched onto Leu⁻/8% glucose plates to increase plasmid copy number.Leu⁻ starter cultures were grown for 24 hours at 30° C. and then diluted1:20 in YEPD (yeast extract bactopeptone 2% glucose) media. The cellswere grown for 48 hours at 30° C. and harvested. To test for expressionof the MEFA 7 recombinant antigen, an aliquot of the cells was lysedwith glass beads in lysis buffer (10 mM Tris-Cl pH 7.5, 1 mM EDTA, 10 mMDTT). The lysate was centrifuged at high speed. The supernatant andinsoluble pellet were analyzed on SDS protein gels. MEFA 7 was highlyenriched in the insoluble pellet fraction.

The MEFA 7 antigen was purified as follows. S. cerevisiae cellsexpressing MEFA 7 were harvested as described above. The cells weresuspended in lysis buffer (50 mM Tris, 0.15 M NaCl, 1 mM EDTA, 1 mMPMSF, pH 8.0) and lysed in a Dyno-Mill (Wab Willy A. Bachofon, Basel,Switzerland) or equivalent apparatus using glass beads. The lysate wascentrifuged at low speed conditions (3,000 to 5,000 rpm, 15 min) and thepellet containing the insoluble protein fraction was washed withincreasing concentrations of urea (1 M, 2 M, 3 M) in lysis buffer.Protein was solubilized from the centrifugation pellet with 0.1 N NaOH,4 M urea in lysis buffer. Cell debris was removed by low speedcentrifugation at 3,000 to 5,000 rpm, 15 min. The supernatant wasadjusted to pH 8.0 with 6 N HCl to precipitate proteins insoluble underthese conditions.

The precipitate was removed by centrifugation and the supernatant wasadjusted to 2.3% SDS, 50 mM DTT, pH 8.0 and boiled for 3 min. Proteinsin the mixture were fractionated by gel filtration on a PharmaciaSephacryl S-400 in phosphate buffered saline containing 0.1% SDS, 1 mMEDTA and adjusted to pH 7.4. Column eluate fractions containing MEFA 7were collected, pooled, and concentrated on an Amicon YM-30 membrane.Gel filtration was repeated on the pooled fractions using the samecolumn and conditions.

During the analysis of MEFA 7 in a trial assay, it was discovered that amonoclonal antibody used as a detection conjugate reacted with aspecific sequence of the core epitope (amino acids 33-38). Thus,ps.MEFA7.1 was designed to eliminate amino acids 33-38 from the coreepitope region.

A yeast expression vector for MEFA 7.1 was made as follows. First, thedouble repeat of the core epitope at the 3′ end of ps.MEFA7 wasmodified. To do so, an NcoI/KpnI synthetic fragment of 206 bp, encodingthe first core epitope repeat (amino acids 9-32, 39-42 and 64-88 ofHCV-1, and amino acids 67-82 of HCV-2), and a KpnI/SalI syntheticfragment of 233 bp encoding amino acids 83 and 84 of HCV-2, followed bythe second core epitope repeat (amino acids 9-32, amino acids 39-42 andamino acids 64-88, HCV-1, amino acids 67-84, HCV-2) were subclonedrespectively into a pSP72new.NcoI/KpnI vector and a pSP72new.KpnI/SalIvector. After HB101 transformation, clone identification and sequenceconfirmation, pSP72newNKi clone #21 was digested with NcoI and KpnI toisolate the NcoI/KpnI fragment of 206 bp and pSP72newKSi clone #32 wasdigested with KpnI and SalI to isolate the KpnI/SalI fragment of 233 bp.

Plasmid ps.MEFA7.1 was assembled by ligating the following fragmentsinto the pBS24.1 BamHI/SalI yeast expression vector (see, FIG. 8):

the BamHI/HindIII fragment of 1896 bp, described above for ps.MEFA7;

the HindIII/SphI fragment of 1560 bp described above for ps.MEFA7;

an SphI/NcoI fragment of 776 bp isolated from ps.MEFA7 encoding the5-1-1 epitopes, c100 epitope and NS5 epitope;

the NcoI/KpnI fragment of 206 bp;

and KpnI/SalI fragment of 233 bp.

S. cerevisiae strain AD3 was transformed with ps.MEFA7.1 and singletransformants were checked for expression after depletion of glucose inthe medium, as described above. The recombinant protein was expressed athigh levels in yeast, as detected by Coomassie blue staining.

The MEFA 7.1 antigen was purified as follows. S. cerevisiae cellsexpressing MEFA 7.1 were harvested as described above. The cells weresuspended in lysis buffer (50 mM Tris, 0.15 M NaCl, 1 mM EDTA, pH 8.0)and lysed in a Dyno-Mill (Wab Willy A. Bachofen, Basel, Switzerland) orequivalent apparatus using glass beads. The lysate was centrifuged at10,000 rpm, 30 min in a JA-10 rotor, and the pellet containing theinsoluble protein fraction was washed with increasing concentrations ofUrea (1 M, 2 M, 3 M) in lysis buffer. Protein was solubilized from thecentrifugation pellet with 0.1 N NaOH, 4 M Urea, 50 mM DTT in lysisbuffer. Cell debris was removed by centrifugation at 14,000 rpm, 20 minin a JA-14 rotor. The supernatant was adjusted to pH 8.0 with 6 N HCl toprecipitate proteins insoluble under these conditions.

The precipitate was removed by centrifugation at 14,000 rpm, 20 min in aJA-14 rotor. The supernatant was adjusted to 2.3% SDS and heated to70-75° C. in boiling water then cooled to room temperature. Proteins inthe mixture were fractionated by gel filtration on a Pharmacia SephacrylS-400 HR in PBS containing 0.1% SDS, 1 mM EDTA and adjusted to pH 7.4.Column eluate fractions containing MEFA 7.1 were collected, pooled, andconcentrated on an Amicon YM-30 membrane. Pooled gel filtrationfractions were adjusted to 2.3% SDS, 50 mM DTT, and heated/cooled asabove. This pool was subjected to a second gel filtration step on aPharmacia Sephacryl S-300 HR column under the same conditions as thefirst gel filtration step.

EXAMPLE 2 Recombinant Production of an NS3/4a Conformational Epitope

A conformational epitope of NS3/4a was obtained as follows. This epitopehas the sequence specified in FIGS. 3A through 3D and differs from thenative sequence at positions 403 (amino acid 1428 of the HCV-1full-length sequence) and 404 (amino acid 1429 of the HCV-1 full-lengthsequence). Specifically, the Thr normally occurring at position 1428 ofthe native sequence has been mutated to Pro and Ser which occurs atposition 1429 of the native sequence has been mutated to Ile.

In particular, the yeast expression vector used was pBS24.1, describedabove. Plasmid pd.hcv1a.ns3ns4aPI, which encoded a representative NS3/4aepitope used in the subject immunoassays, was produced as follows. A twostep procedure was used. First, the following DNA pieces were ligatedtogether: (a) synthetic oligonucleotides which would provide a 5′HindIII cloning site, followed by the sequence ACAAAACAAA (SEQ ID NO:6),the initiator ATG, and codons for HCV1a, beginning with amino acid 1027and continuing to a BglI site at amino acid 1046; (b) a 683 bp BglI-ClaIrestriction fragment (encoding amino acids 1046-1274) frompAcHLTns3ns4aPI; and (c) a pSP72 vector (Promega, Madison, Wis.,GenBank/EMBL Accession Number X65332) which had been digested withHindIII and ClaI, dephosphorylated, and gel-purified. PlasmidpAcHLTns3ns4aPI was derived from pAcHLT, a baculovirus expression vectorcommercially available from BD Pharmingen (San Diego, Calif.). Inparticular, a pAcHLT EcoRI-PstI vector was prepared, as well as thefollowing fragments: EcoRI-AlwnI, 935 bp, corresponding to amino acids1027-1336 of the HCV-1 genome; AlwnI-SacII, 247 bp, corresponding toamino acids 1336-1419 of the HCV-1 genome; HinfI-BglI, 175 bp,corresponding to amino acids 1449-1509 of the HCV-1 genome; BglI-PstI,619 bp, corresponding to amino acids 1510-1711 of the HCV-1 genome, plusthe transcription termination codon. A SacII-HinfI syntheticallygenerated fragment of 91 bp, corresponding to amino acids 1420-1448 ofthe HCV-1 genome and containing the PI mutations (Thr-1428 mutated toPro, Ser-1429 mutated to Ile), was ligated with the 175 bp HinfI-BglIfragment and the 619 bp BglI-PstI fragment described above and subclonedinto a pGEM-5Zf(+) vector digested with SacII and PstI. pGEM-5Zf(+) is acommercially available E. coli vector (Promega, Madison, Wis.,GenBank/EMBL Accession Number X65308). After transformation of competentHB101 cells, miniscreen analysis of individual clones and sequenceverification, an 885 bp SacII-PstI fragment from pGEM5.PI clone2 wasgel-purified. This fragment was ligated with the EcoRI-AlwnI 935 bpfragment, the AlwnI-SacII247 bp fragment and the pAcHLT EcoRI-PstIvector, described above. The resultant construct was namedpAcHLTns3ns4aPI.

The ligation mixture above was transformed into HB101-competent cellsand plated on Luria agar plates containing 100 μg/ml ampicillin.Miniprep analyses of individual clones led to the identification ofputative positives, two of which were amplified. The plasmid DNA forpSP72.1aHC, clones #1 and #2 were prepared with a Qiagen Maxiprep kitand were sequenced.

Next, the following fragments were ligated together: (a) a 761 bpHindIII-ClaI fragment from pSP721aHC #1 (pSP72.1aHC was generated byligating together the following: pSP72 which had been digested withHindIII and ClaI, synthetic oligonucleotides which would provide a 5′HindIII cloning site, followed by the sequence ACAAAACAAA (SEQ ID NO:6),the initiation codon ATG, and codons for HCV1a, beginning with aminoacid 1027 and continuing to a BglII site at amino acid 1046, and a 683bp BglII-ClaI restriction fragment (encoding amino acids 1046-1274) frompAcHLTns3ns4aPI); (b) a 1353 bp BamHI-HindIII fragment for the yeasthybrid promoter ADH2/GAPDH; (c) a 1320 bp ClaI-SalI fragment (encodingHCV1a amino acids 1046-1711 with Thr 1428 mutated to Pro and Ser 1429mutated to Ile) from pAcHLTns3ns4aPI; and (d) the pBS24.1 yeastexpression vector which had been digested with BamHI and SalI,dephosphorylated and gel-purified. The ligation mixture was transformedinto competent HB101 and plated on Luria agar plates containing 100μg/ml ampicillin. Miniprep analyses of individual colonies led to theidentification of clones with the expected 3446 bp BamHI-SalI insertwhich was comprised of the ADH2/GAPDH promoter, the initiator codon ATGand HCV1a NS3/4a from amino acids 1027-1711 (shown as amino acids 1-686of FIGS. 3A-3D), with Thr 1428 (amino acid position 403 of FIGS. 3A-3D)mutated to Pro and Ser 1429 (amino acid position 404 of FIGS. 3A-3D)mutated to Ile. The construct was named pd.HCV1a.ns3ns4aPI (see, FIG.9).

S. cerevisiae strain AD3 was transformed with pd.HCV1a.ns3ns4aPI andsingle transformants were checked for expression after depletion ofglucose in the medium. The recombinant protein was expressed at highlevels in yeast, as detected by Coomassie blue staining and confirmed byimmunoblot analysis using a polyclonal antibody to the helicase domainof NS3.

EXAMPLE 3 Purification of NS3/4a Conformational Epitope

The NS3/4a conformational epitope was purified as follows. S. cerevisiaecells from above, expressing the NS3/4a epitope were harvested asdescribed above. The cells were suspended in lysis buffer (50 mM Tris pH8.0, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 0.1 μM pepstatin, 1 μMleupeptin) and lysed in a Dyno-Mill (Wab Willy A. Bachofon, Basel,Switzerland) or equivalent apparatus using glass beads, at a ratio of1:1:1 cells:buffer:0.5 mm glass beads. The lysate was centrifuged at30100×g for 30 min at 4° C. and the pellet containing the insolubleprotein fraction was added to wash buffer (6 ml/g start cell pelletweight) and rocked at room temperature for 15 min. The wash bufferconsisted of 50 mM NaPO₄ pH 8.0, 0.3 M NaCl, 5 mM β-mercaptoethanol, 10%glycerol, 0.05% octyl glucoside, 1 mM EDTA, 1 mM PMSF, 0.1 μM pepstatin,1 μM leupeptin. Cell debris was removed by centrifugation at 30100×g for30 min at 4° C. The supernatant was discarded and the pellet retained.

Protein was extracted from the pellet as follows. 6 ml/g extractionbuffer was added and rocked at room temperature for 15 min. Theextraction buffer consisted of 50 mM Tris pH 8.0, 1 M NaCl, 5 mMβ-mercaptoethanol, 10% glycerol, 1 mM EDTA, 1 mM PMSF, 0.1 μM pepstatin,1 μM leupeptin. This was centrifuged at 30100×g for 30 min at 4° C. Thesupernatant was retained and ammonium sulfate added to 17.5% using thefollowing formula: volume of supernatant (ml) multiplied by x % ammoniumsulfate/(1−x % ammonium sulfate)=ml of 4.1 M saturated ammonium sulfateto add to the supernatant. The ammonium sulfate was added dropwise whilestirring on ice and the solution stirred on ice for 10 min. The solutionwas centrifuged at 17700×g for 30 min at 4° C. and the pellet retainedand stored at 2° C. to 8° C. for up to 48 hrs.

The pellet was resuspended and run on a Poly U column (Poly U Sepharose4B, Amersham Pharmacia) at 4° C. as follows. Pellet was resuspended in 6ml Poly U equilibration buffer per gram of pellet weight. Theequilibration buffer consisted of 25 mM HEPES pH 8.0, 200 mM NaCl, 5 mMDTT (added fresh), 10% glycerol, 1.2 octyl glucoside. The solution wasrocked at 4° C. for 15 min and centrifuged at 31000×g for 30 min at 4°C.

A Poly U column (1 ml resin per gram start pellet weight) was prepared.Linear flow rate was 60 cm/hr and packing flow rate was 133% of 60cm/hr. The column was equilibrated with equilibration buffer and thesupernatant of the resuspended ammonium sulfate pellet was loaded ontothe equilibrated column. The column was washed to baseline with theequilibration buffer and protein eluted with a step elution in thefollowing Poly U elution buffer: 25 mM HEPES pH 8.0, 1 M NaCl, 5 mM DTT(added fresh), 10% glycerol, 1.2 octyl glucoside. Column eluate was runon SDS-PAGE (Coomassie stained) and aliquots frozen and stored at −80°C. The presence of the NS3/4a epitope was confirmed by Western blot,using a polyclonal antibody directed against the NS3 protease domain anda monoclonal antibody against the 5-1-1 epitope (HCV 4a).

Additionally, protease enzyme activity was monitored during purificationas follows. An NS4A peptide (KKGSVIVGRIVLSGKPAIIPKK (SEQ ID NO:7)), andthe sample containing the NS3/4a conformational epitope, were diluted in90 μl of reaction buffer (25 mM Tris, pH 7.5, 0.15M NaCl, 0.5 mM EDTA,10% glycerol, 0.05 n-Dodecyl B-D-Maltoside, 5 mM DTT) and allowed to mixfor 30 minutes at room temperature. 90 μl of the mixture were added to amicrotiter plate (Costar, Inc., Corning, N.Y.) and 10 μl of HCVsubstrate (AnaSpec, Inc., San Jose Calif.) was added. The plate wasmixed and read on a Fluostar plate reader. Results were expressed asrelative fluorescence units (RFU) per minute.

Using these methods, the product of the 1 M NaCl extraction contained3.7 RFU/min activity, the ammonium sulfate precipitate had an activityof 7.5 RFU/min and the product of the Poly U purification had anactivity of 18.5 RFU/min.

EXAMPLE 4 Coating Solid Support with the HCV Antigens

The HCV NS3/4a conformational epitope and MEFA 7.1 antigen were coatedonto plates as follows. HCV coating buffer (50 mM NaPO4 pH 7.0, 2 mMEDTA and 0.1% Chloroacetamide) was filtered through a 0.22μ filter unit.The following reagents were then added sequentially to the HCV coatingbuffer and stirred after each addition: 2 μg/ml BSA-Sulfhydryl Modified,from a 10 mg/ml solution (Bayer Corp. Pentex, Kankakee, Ill.); 5 mM DTTfrom a 1 M solution (Sigma, St. Louis, Mo.); 0.45 μg/ml NS3/4a (proteinconcentration of 0.3 mg/ml); 0.375 μg/ml MEFA 7.1 (protein concentrationof 1 mg/ml). The final solution was stirred for 15 minutes at roomtemperature.

200 μl of the above solution was added to each well of a Costar highbinding, flat bottom plate (Corning Inc., Corning, N.Y.) and the plateswere incubated overnight in a moisture chamber. The plates were thenwashed with wash buffer (1× PBS, 0.1% TWEEN-20), Tapped dry and 285 μlOrtho Post-Coat Buffer (1× PBS, pH 7.4, 1% BSA, 3% sucrose) added. Theplates were incubated for at least 1 hour, tapped and dried overnight at2-8° C. The plates were pouched with desiccants for future use.

EXAMPLE 5 Early Seroconversion Studies

The performance of the NS3/4a and MEFA 7.1 antigens in a combinationassay (HCV 4.0) was compared to other HCV assays to test theseroconversion detection limits and to compare these limits to thoseobtained in other commercially available assays. Panels of commerciallyavailable human blood samples were used which were HCV-infected. The PHVpanels shown in the tables below were purchased from Boston Biomedica,Inc., West Bridgewater, Mass. (BBI). The 6212 panels were purchased fromBioclinical Partners, Franklin, Mass. (BCP). The SC panels werepurchased from North American Biologics, Inc., BocoRatan, Fla. (NABI).The day on which the blood samples was obtained is indicated in thetables.

The HCV 4.0 assay was conducted as follows. 200 μl of specimen diluentbuffer (1 g/l casein, 100 mg/l recombinant human SOD, 1 g/lchloracetamide, 10 g/l BSA, 500 mg/l yeast extract, 0.366 g/l EDTA,1.162 g/l KPO₄, 5 ml/l Tween-20, 29.22 g/l NaCl, 1.627 g/l NaPO₄, 1%SDS) was added to the coated plates. 20 μl of sample was then added.This was incubated at 37° C. for one hour. The plates were washed withwash buffer (1× PBS, pH 7.4, 0.1% Tween-20). 200 μl conjugate solution(a mouse anti-human IgG-HRP, such as mouse anti-human IgG-HRP diluted1:22,000 in ORTHO HCV 3.0 ELISA Test System with Enhanced SAVe bulkconjugate diluent (Ortho-Clinical Diagnostics, Raritan, N.J.) was addedand incubated for 60 minutes at 37° C. This was washed as above, and 200μl substrate solution (1 OPD tablet/10 ml) was added. The OPD tabletcontains o-phenylenediamine dihydrochloride and hydrogen peroxide forhorse radish peroxidase reaction color development. This was incubatedfor 30 minutes at room temperature in the dark. The reaction was stoppedby addition of 50 μl 4N H₂SO₄ and the plates were read at 492 nm,relative to absorbance at 690 nm as control.

The other assays used in the study were as follows:

The Abbott PRISM assay (Abbott Laboratories, Abbott Park, Ill.), iscommercially available and is an antibody-based detection assay. Theassay was performed using the manufacturer's instructions.

The ORTHO HCV Version 3.0 ELISA Test System (HCV 3.0) (Ortho ClinicalDiagnostics, Raritan, N.J.) is an antibody-based detection assay. Theassay was conducted using the manufacturer's instructions.

The Pasteur MONOLISA anti-HCV Plus Version 2 assay (Sanofi DiagnosticsPasteur, Marnes-1a-Coquette, France) is an antibody-based detectionassay. The assay was performed using the manufacturer's instructions.

The performance of the HCV 4.0 assay was compared to the HCV 3.0, PRISMand Pasteur assays (see, Tables 3 and 4). HCV antibodies present in theblood panels (anti-c33c or anti-c22) are set forth in Table 4. Inparticular, 17 seroconversion panels from the three commercial sourcesset forth above were assayed using the techniques above. As can be seen,for the c33c panels, HCV 4.0 showed earlier detection (by 1-3 bleeds)than HCV 3.0 in 9 out of 9 c33c panels, and earlier detection than PRISMin 6 out of 9 panels and equivalent detection as compared with PRISM in3 of the 9 panels. For the c22 panels, HCV 4.0 showed earlier detectionthan HCV 3.0 in 3 of 8 panels and equivalent detection in the other 5panels. HCV 4.0 also showed earlier detection than PRISM in 2 of 8panels and equivalent detection in 6 of 8 panels. The range ofimprovement seen was 2-14 days over both the HCV 3.0 and PRISM assays.

TABLE 3 Ortho Abbott HCV 4.0 HCV 3.0 Prism ID Bleed Day s s/co s/co s/coPHV 904-1  0 0.031 0.05 0.01 0.12 PHV 904-2  2 0.024 0.04 0.01 0.08 PHV904-3  7 1.391 2.11 0.33 0.51 PHV 904-4  9 2.813 4.28 1.10 1.56 PHV904-5  14 3.197 4.66 3.27 3.54 PHV 904-6  21 3.176 4.83 3.92 4.45 PHV904-7  23 3.554 5.40 4.26 4.69 PHV 905-1  0 0.015 0.02 0.02 0.06 PHV905-2  4 0.019 0.03 0.01 0.07 PHV 905-3  7 0.079 0.12 0.02 0.14 PHV905-4  11 0.950 1.44 0.42 0.70 PHV 905-5  14 1.586 2.41 0.80- 1.21 PHV905-6  18 2.529 3.84 1.32 2.00 PHV 905-7  21 3.177 4.83 2.54 3.44 PHV905-8  25 3.419 5.20 4.45 4.74 PHV 905-9  28 3.408 5.18 4.85 5.04 PHV907-1  0 0.030 0.05 0.01 0.09 PHV 907-2  4 0.023 0.03 0.01 0.09 PHV907-3  7 0.021 0.03 0.01 0.11 PHV 907-4  13 0.148 0.22 0.13 0.35 PHV907-5  18 1.726 2.62 0.83- 1.68 PHV 907-6  21 2.785 4.23 1.59 3.12 PHV907-7 164 3.279 4.98 4.85 5.16 PHV 908-1  0 0.029 0.04 0.01 0.07 PHV908-2  3 0.079 0.12 0.01 0.08 PHV 908-3  5 0.399 0.61 0.01 0.13 PHV908-4  11 1.780 2.71 0.50 1.27 PHV 908-5  13 2.068 3.14 0.67 1.61 PHV908-6  19 2.793 4.24 1.16 3.11 PHV 908-7  25 3.390 5.15 3.11 4.29 PHV908-8  27 3.299 5.01 3.78 4.44 PHV 908-9  32 3.474 5.28 4.85 4.54 PHV908-10  35 3.707 5.63 4.85 4.92 PHV 908-11  41 3.363 5.11 4.85 6.09 PHV908-12  45 3.372 5.12 3.80 5.79 PHV 908-13  48 3.278 4.98 4.85 5.56 PHV913-1  0 0.060 0.09 0.01 0.08 PHV 913-2  2 0.242 0.37 0.02 0.10 PHV913-3  7 0.893 1.36 0.43- 0.5- PHV 913-4  9 1.141 1.73 0.54- 0.59- PHV914-1  0 0.033 0.05 0.00 0.06 PHV 914-2  5 0.024 0.04 0.01 0.06 PHV914-3  9 0.135 0.21 0.01 0.06 PHV 914-4  12 2.653 4.03 0.04 0.09 PHV914-5  16 3.020 4.59 0.33- 0.47- PHV 914-6  19 2.302 3.50 0.82- 0.9- PHV914-7  24 2.697 4.10 3.10 2.41 PHV 914-8  30 2.744 4.17 4.85 4.09 PHV914-9  33 2.991 4.55 4.85 4.52 6212-1 Nov. 16, 1995 0.723 1.10 0.01 0.086212-2 Nov. 28, 1995 2.716 4.13 0.85 1.26 6212-3 Nov. 30, 1995 3.1174.74 1.15 1.11 6212-4 Dec. 9, 1995 3.278 4.98 4.08 2.65 6212-5 Dec. 12,1995 3.527 5.36 5.04 3.13 6212-6 Dec. 18, 1995 3.292 5.00 5.51 3.076212-7 Dec. 23, 1995 3.096 4.71 5.43 2.80 6212-8 Jan. 08, 1996 3.2414.93 13.4 3.48 6212-9 Jan. 10, 1996 3.306 5.02 13.4 4.04 6213-1 Jan. 16,1996 0.035 0.05 0.01 0.09 6213-2 Jan. 18, 1996 0.028 0.04 0.00 0.076213-3 Jan. 24, 1996 0.037 0.06 0.01 0.08 6213-4 Jan. 27, 1996 0.0470.07 0.01 0.09 6213-5 Jan. 31, 1996 0.034 0.05 0.01 0.07 6213-6 Feb. 03,1996 0.037 0.06 0.01 0.07 6213-7 Feb. 13, 1996 0.056 0.09 0.01 0.076213-8 Feb. 15, 1996 0.027 0.04 0.01 0.06 6213-9 Feb. 20, 1996 0.0980.15 0.01 0.11 6213-10 Feb. 22, 1996 1.572 2.39 0.46- 1.26 6213-11 Feb.28, 1996 3.410 5.18 4.18 4.75 6213-12 Mar. 02, 1996 3.224 4.90 4.67 4.686214-1 Jan. 13, 1996 0.042 0.06 0.01 0.09 6214-2 Jan. 15, 1996 0.0160.02 0.00 0.07 6214-3 Jan. 21, 1996 0.035 0.05 0.00 0.07 6214-4 Jan. 23,1996 0.028 0.04 0.00 0.07 6214-5 Jan. 29, 1996 0.031 0.05 0.00 0.086214-6 Jan. 31, 1996 0.028 0.04 0.00 0.09 6214-7 Feb. 05, 1996 0.3640.55 0.01 0.59 6214-8 Feb. 07, 1996 0.916 1.39 0.02- 1.36 6214-9 Feb.12, 1996 2.290 3.48 0.94- 2.31 6214-10 Feb. 14, 1996 3.669 5.58 2.293.05 6214-11 Mar. 02, 1996 3.523 5.35 3.15 5.32 6214-12 Mar. 06, 19963.125 4.75 4.75 5.38 6214-13 Mar. 09, 1996 3.246 4.93 4.89 5.17 6222-1Aug. 18, 1996 0.023 0.03 0.01 0.08 6222-2 Aug. 20, 1996 0.151 0.23 0.000.06 6222-3 Sept. 04, 1996 0.053 0.08 0.00 0.06 6222-4 Sept. 06, 19960.016 0.02 0.00 0.06 6222-5 Sept. 11, 1996 0.015 0.02 0.00 0.07 6222-6Sept. 13, 1996 0.009 0.01 0.00 0.06 6222-7 Sept. 23, 1996 0.817 1.240.04 0.36 6222-8 Sept. 27, 1996 2.862 4.35 1.10 3.74 SC-0030-A  1 0.0240.04 0.01 0.05 SC-0030-B  40 1.658 2.52 0.94- 0.54- SC-0030-C  45 2.3723.60 3.07 3.15 SC-0040-A  1 0.773 1.17 0.02 0.09 SC-0040-B  3 1.491 2.270.12 0.54 SC-0040-C  8 2.400 3.65 0.77- 2.66 SC-0040-D  10 2.639 4.011.37 3.23 SC-0040-E  15 3.423 5.20 3.46 3.46

TABLE 4 Bleed days earlier detection Abbott early c33c early Panel HCV3.0 Prism panels c22 panels #1 c33c PHV 904 2 2 PHV 904 PHV 907 #2 c33cPHV 905 7 3 PHV 905 PHV 909 #3 c22 PHV 907 3 0 PHV 908 PHV 910 #4 c33cPHV 908 8 0 PHV 914 PHV 911 #5 c22 PHV 909 0 0 6212 PHV 912 #6 c22 PHV910 0 0 6213 PHV 913 #7 c22 PHV 911 0 0 6214 SC-0010 #8 c22 PHV 912 0 06222 SC-0030 #9 c22 PHV 913 2 2 SC-0040 #10 c33c PHV 914 12 12 #11 c33c6212 14 12 #12 c33c 6213 6 0 #13 c33c 6214 7 0 #14 c33c 6222 4 4 #15 c22SC-0010 0 0 #16 c22 SC-0030 5 5 #17 c33c SC-0040 9 7 Range ofImprovement 2-14 days 2-12 days

EXAMPLE 6 HCV 4.0 Genotype Sensitivity

The genotype sensitivity of the HCV 4.0 assay was compared to the HCV3.0 and Pasteur assays, described above. In particular, samples from 10different HCV genotypes, specified in Table 5, were diluted as indicatedin the table (2-fold or 10-fold depending on initial sample titering)and used in the three assays, using the procedures described above. Allthree tests were run simultaneously. The data is shown as signal or rawO.D. The data suggests that the HCV 4.0 prototype is more sensitive indetecting diluted genotype samples.

TABLE 5 HCV 4.0 and Genotype Dilutional Sensitivity HCV 4.0 HCV 3.0Monolisa Ver. 2 HCV 4.0 HCV 3.0 Monolisa Ver. 2 prototype Ortho Pasteurprototype Ortho Pasteur dilution genotype s s s genotype s s s 1:2500 1b2.866 1.007 0.694 4b/c 2.478 0.701 0.551 1:5000 2.074 0.393 0.218 1.1250.256 0.195 1:10000 1.099 0.159 0.084 0.609 0.087 0.076 1:20000 0.4030.045 0.028 0.216 0.035 0.033 1:2500 2b 1.430 0.295 0.658 4a 2.831 0.6320.462 1:5000 0.551 0.108 0.207 1.752 0.193 0.181 1:10000 0.225 0.0320.061 0.717 0.069 0.076 1:20000 0.074 0.010 0.019 0.248 0.015 0.0251:2500 2a/c 1.952 0.467 1.653 4c 1.751 0.457 1.147 1:5000 0.917 0.1360.782 0.856 0.169 0.474 1:10000 0.395 0.049 0.286 0.384 0.055 0.1781:20000 0.108 0.011 0.105 0.141 0.018 0.058 1:2500 3a 2.580 0.514 0.9415a 2.682 1.496 2.271 1:5000 1.622 0.218 0.353 2.744 0.827 0.988 1:100000.873 0.067 0.164 1.587 0.316 0.395 1:20000 0.398 0.023 0.050 0.7260.097 0.120 1:2500 3e 1.207 0.158 0.291 1:10 6 3.516 3.247 ND 1:50000.461 0.039 0.114 1:100 3.602 3.594 ND 1:10000 0.155 0.011 0.053 1:10003.224 2.863 ND 1:20000 0.054 0.003 0.024 1:10000 1.192 0.380 ND

EXAMPLE 7 Competition Studies

The following competition study was conducted in order to assess whetherthe NS3/4a conformational epitope detected different antibodies thanother HCV antigens. In particular, the NS3/4a antigen was compared withthe c200 antigen as follows.

0.5 μg and 1.0 μg of NS3/4a, produced as described above, or c200(Hepatology (1992) 15:19-25, available in the ORTHO HCV Version 3.0ELISA Test System, Ortho-Clinical Diagnostics, Raritan, N.J.), weremixed with 20 μl of sample PHV914-5 (an early seroconversion bleedobtained from blood of an infected individual) in a total volume of 220μl (1× PBS). The mixture was incubated for 1 hour in microwells at 37°C. The mixture was then transferred to NS3/4a-coated plates andincubated for 1 hour at 37° C. Plates were washed and assayed asfollows.

1 μg of c200 antigen was added to 10 μl of sample PHV914-5 in a totalvolume of about 220 μl. The mixture was incubated for 1 hour in a microwell at 37° C. and 200 μl transferred to an NS3/4a-coated plate (100ng/assay) and incubated for 1 hour at 37° C. Plates were washed fivetimes with 1× PBS, 0.1% Tween-20. 200 μl of conjugate solution(described above) were added, and the plates incubated and assayed asdescribed in Example 4 for the HCV 4.0 assay. Controls which consistedof PHV914-5 and 1× PBS (without antigen) were also treated as above.

Results are shown in Table 6. Percent inhibition results shown in column4 are calculated as column 3 minus (column 2 divided by column 3 times100). As can be seen, the data show that NS34a is neutralized by earlyseroconversion antibodies and c200 is not. A strong signal was achievedwhen antibodies in PHV914-5 c33c early seroconversion panel memberreacted with the NS34a coated on the plate. The c200 antigen was notneutralized by these antibodies. This is shown in the top panel of Table6. When NS34a was mixed with the PHV914-5 sample, it was neutralized andtherefore no antibodies were present in the sample to react with NS34athat was coated on the microplate. The data indicate that NS34a may bedetecting a different class of antibodies than is detected by c200.

TABLE 6 Competition Studies to Show NS34a Antigen Detects DifferentAntibodies in Early c33c Seroconversion Panel Compared to c200 Antigen 32 *Control 1 PHV914-5 1xPBS 4 c200 + s s % Inhibition   1 ug 1.450 1.64512   1 ug 1.545 1.687 8 0.5 ug 1.557 1.913 19 0.5 ug 1.719 1.804 5PHV914-5 NS3/4a + s s   1 ug 0.054 1.599 97   1 ug 0.037 1.677 98 0.5 ug0.066 1.672 96 0.5 ug NA 1.524 NA

EXAMPLE 8 Stability Studies of NS3/4a Conformational Epitope

To assess the role of stability of the NS3/4a epitope to assayperformance, the following study was done to determine NS3/4aimmunoreactivity versus time at room temperature. Small aliquots ofstock NS3/4a were allowed to sit at room temperature and then frozen atintervals as shown in Table 7. All vials were coated simultaneously andtested against two early NS3 seroconversion panels. Assays wereconducted as described above in Example 5 for HCV 4.0.

As can be seen in Table 7, the NS3/4a stock is not stable andimmunoreactivity decreases with time. In addition, maintaining NS3/4aconformation is necessary for immunoreactivity.

Further stability studies were conducted as follows. Two conformationalmonoclonal antibodies made against NS3/4a using standard procedures weresubstituted for anti-HCV early seroconversion panels. Stock NS3/4a vialswere stored at room temperature at time intervals 3, 6 and 24 hours. TheNS3/4a from the frozen vials was coated at 90 ng/ml and assayed usingthe procedure described above. Results suggested that the twomonoclonals were indeed conformational and their reactivity wassensitive to the handling of stock NS3/4a antigen at room temperature.The reactivity of a positive control monoclonal antibody did not change.

TABLE 7 Time (hrs) 0 6 21.4 29 35.5 46 52 control A D G H I K N Referencs/co s/co s/co s/co s/co s/co s/co s/co PHV 0.0 0.0 0.0 0.0 0.0 0.0 0.00.0 904-1 PHV 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 904-2 PHV 1.5 0.3 0.1 0.00.0 0.0 0.0 1.8 904-3 PHV 3.7 1.0 0.2 0.1 0.1 0.1 0.1 4.4 904-4 PHV 4.82.0 0.7 0.6 0.3 0.2 0.3 5.5 904-5 PHV 5.4 2.8 1.1 1.0 0.6 0.5 0.6 5.8904-6 PHV 5.1 3.4 1.5 1.0 1.1 0.5 0.7 5.4 904-7 PHV 0.0 0.0 0.0 0.0 0.00.0 0.0 0.0 914-1 PHV 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 914-2 PHV 0.0 0.00.0 0.0 0.0 0.0 0.0 0.0 914-3 PHV 0.5 0.1 0.0 0.0 0.0 0.0 0.0 0.7 914-4PHV 2.1 0.4 0.0 0.0 0.0 0.0 0.0 3.0 914-5 PHV 2.3 0.4 0.0 0.0 0.0 0.00.0 3.4 914-6 PHV 2.8 0.5 0.1 0.1 0.0 0.0 0.0 4.9 914-7 PHV 2.9 0.7 0.10.1 0.1 0.1 0.1 4.9 914-8 Enzyme RFU/min 8.75 4.14 3.08 1.88 1.75 1.750.75

EXAMPLE 9 Immunoreactivity of NS3/4a Conformational Epitope VerusDenatured NS3/4a

The immunoreactivity of the NS3/4a conformational epitope, produced asdescribed above, was compared to NS3/4a which had been denatured byadding SDS to the NS3/4a conformational epitope preparation to a finalconcentration of 2%. The denatured NS3/4a and conformational NS3/4a werecoated onto microtiter plates as described above. The c200 antigen(Hepatology (1992) 15:19-25, available in the ORTHO HCV Version 3.0ELISA Test System, Ortho-Clinical Diagnostics, Raritan, N.J.) was alsocoated onto microtiter plates. The c200 antigen was used as a comparisonit is presumed to be non-conformational due to the presence of reducingagent (DTT) and detergent (SDS) in its formulation.

The immunoreactivity was tested against two early HCV seroconversionpanels, PIV 904 and PHV 914 (commercially available human blood samplesfrom Boston Biomedica, Inc., West Bridgewater, Mass.), using the ELISAassay procedure described above. The results are shown in Table 8. Thedata suggests that the denatured or linearized form of NS3/4a (as wellas c200) does not detect early seroconversion panels as early as theNS3/4a conformational epitope.

TABLE 8 NS3/4a vs. denatured NS3/4a *Spiked 2% SDS to stock NS3/4aNS3/4a dNS3/4a* c200 NS3/4a dNS3/4a* c200 OD OD OD s/co s/co s/co HCVPHV 904-1 0.012 0.012 0.009 0.02 0.02 0.01 Seroconversions PHV 904-20.011 0.009 0.008 0.02 0.01 0.01 PHV 904-3 1.124 0.071 0.045 1.80 0.110.07 PHV 904-4 2.401 0.273 0.129 3.85 0.44 0.21 PHV 904-5 3.022 0.7930.347 4.85 1.28 0.57 PHV 904-6 2.711 1.472 0.774 4.35 2.37 1.28 PHV904-7 3.294 1.860 0.943 5.28 2.99 1.55 PHV 914-1 0.006 0.004 0.001 0.010.01 0.00 PHV 914-2 0.005 0.004 0.002 0.01 0.01 0.00 PHV 914-3 0.0980.003 0.001 0.16 0.00 0.00 PHV 914-4 1.118 0.006 0.004 1.79 0.01 0.01PHV 914-5 2.035 0.044 0.022 3.26 0.07 0.04 PHV 914-6 2.092 0.074 0.0253.35 0.12 0.04 PHV 914-7 2.519 0.281 0.132 4.04 0.45 0.22 PHV 914-82.746 0.907 0.500 4.40 1.46 0.82 PHV 914-9 3.084 1.730 0.931 4.94 2.781.53 HCV 3.0 Neg.Cont. 0.023 0.024 0.008 Controls Neg.Cont. 0.027 0.0240.007 Neg.Cont. 0.021 0.017 0.005 average 0.024 0.022 0.007 cutoff 0.6240.622 0.607 Pos. Cont. 1.239 0.903 0.575 1.99 1.45 0.95 Pos. Cont. 1.4450.916 0.614 2.32 1.47 1.01

Immunoreactivity of the conformational epitope was also tested usingmonoclonal antibodies to NS3/4a, made using standard procedures. Thesemonoclonal antibodies were then tested in the ELISA format describedabove against NS3/4a and denatured NS3/4a and c200 antigen. The datashow that anti-NS3/4a monoclonals react to the NS3/4a and denaturedNS3/4a in a similar manner to the seroconversion panels shown in Table9. This result also provides further evidence that the NS3/4a isconformational in nature as monoclonal antibodies can be made which aresimilar in reactivity to the early c33c seroconversion panels.

TABLE 9 Plate NS3/4a dNS3/4a c200 Monoclonal OD OD OD 4B9/E3 1:100 1.8200.616 0.369 1:1000 1.397 0.380 0.246 1:10000 0.864 0.173 0.070 1:200000.607 0.116 0.085 5B7/D7 1:100 2.885 0.898 0.436 1:1000 2.866 0.5410.267 1:10000 1.672 0.215 0.086 1:20000 1.053 0.124 0.059 1A8/H2 1:1001.020 0.169 0.080 1:1000 0.921 0.101 0.043 1:10000 0.653 0.037 0.0131:20000 0.337 0.027 0.011

Accordingly, novel HCV detection assays have been disclosed. From theforegoing, it will be appreciated that, although specific embodiments ofthe invention have been described herein for purposes of illustration,various modifications may be made without deviating from the spirit andscope thereof.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 7 <210> SEQ ID NO 1 <211> LENGTH: 2058<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:      representative NS3/4a conformational an #tigen <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)..(2058) <400> SEQUENCE: 1atg gcg ccc atc acg gcg tac gcc cag cag ac#a agg ggc ctc cta ggg       48Met Ala Pro Ile Thr Ala Tyr Ala Gln Gln Th #r Arg Gly Leu Leu Gly1               5    #                10   #                15tgc ata atc acc agc cta act ggc cgg gac aa#a aac caa gtg gag ggt       96Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Ly #s Asn Gln Val Glu Gly            20       #            25       #            30gag gtc cag att gtg tca act gct gcc caa ac#c ttc ctg gca acg tgc      144Glu Val Gln Ile Val Ser Thr Ala Ala Gln Th #r Phe Leu Ala Thr Cys        35           #        40           #        45atc aat ggg gtg tgc tgg act gtc tac cac gg#g gcc gga acg agg acc      192Ile Asn Gly Val Cys Trp Thr Val Tyr His Gl #y Ala Gly Thr Arg Thr    50               #    55               #    60atc gcg tca ccc aag ggt cct gtc atc cag at#g tat acc aat gta gac      240Ile Ala Ser Pro Lys Gly Pro Val Ile Gln Me #t Tyr Thr Asn Val Asp65                   #70                   #75                   #80caa gac ctt gtg ggc tgg ccc gct ccg caa gg#t agc cga tca ttg aca      288Gln Asp Leu Val Gly Trp Pro Ala Pro Gln Gl #y Ser Arg Ser Leu Thr                85   #                90   #                95ccc tgc act tgc ggc tcc tcg gac ctt tac ct#g gtc acg agg cac gcc      336Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Le #u Val Thr Arg His Ala            100       #           105       #           110gat gtc att ccc gtg cgc cgg cgg ggt gat ag#c agg ggc agc ctg ctg      384Asp Val Ile Pro Val Arg Arg Arg Gly Asp Se #r Arg Gly Ser Leu Leu        115           #       120           #       125tcg ccc cgg ccc att tcc tac ttg aaa ggc tc#c tcg ggg ggt ccg ctg      432Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly Se #r Ser Gly Gly Pro Leu    130               #   135               #   140ttg tgc ccc gcg ggg cac gcc gtg ggc ata tt#t agg gcc gcg gtg tgc      480Leu Cys Pro Ala Gly His Ala Val Gly Ile Ph #e Arg Ala Ala Val Cys145                 1 #50                 1 #55                 1 #60acc cgt gga gtg gct aag gcg gtg gac ttt at#c cct gtg gag aac cta      528Thr Arg Gly Val Ala Lys Ala Val Asp Phe Il #e Pro Val Glu Asn Leu                165   #               170   #               175gag aca acc atg agg tcc ccg gtg ttc acg ga#t aac tcc tct cca cca      576Glu Thr Thr Met Arg Ser Pro Val Phe Thr As #p Asn Ser Ser Pro Pro            180       #           185       #           190gta gtg ccc cag agc ttc cag gtg gct cac ct#c cat gct ccc aca ggc      624Val Val Pro Gln Ser Phe Gln Val Ala His Le #u His Ala Pro Thr Gly        195           #       200           #       205agc ggc aaa agc acc aag gtc ccg gct gca ta#t gca gct cag ggc tat      672Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Ty #r Ala Ala Gln Gly Tyr    210               #   215               #   220aag gtg cta gta ctc aac ccc tct gtt gct gc#a aca ctg ggc ttt ggt      720Lys Val Leu Val Leu Asn Pro Ser Val Ala Al #a Thr Leu Gly Phe Gly225                 2 #30                 2 #35                 2 #40gct tac atg tcc aag gct cat ggg atc gat cc#t aac atc agg acc ggg      768Ala Tyr Met Ser Lys Ala His Gly Ile Asp Pr #o Asn Ile Arg Thr Gly                245   #               250   #               255gtg aga aca att acc act ggc agc ccc atc ac#g tac tcc acc tac ggc      816Val Arg Thr Ile Thr Thr Gly Ser Pro Ile Th #r Tyr Ser Thr Tyr Gly            260       #           265       #           270aag ttc ctt gcc gac ggc ggg tgc tcg ggg gg#c gct tat gac ata ata      864Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gl #y Ala Tyr Asp Ile Ile        275           #       280           #       285att tgt gac gag tgc cac tcc acg gat gcc ac#a tcc atc ttg ggc att      912Ile Cys Asp Glu Cys His Ser Thr Asp Ala Th #r Ser Ile Leu Gly Ile    290               #   295               #   300ggc act gtc ctt gac caa gca gag act gcg gg#g gcg aga ctg gtt gtg      960Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gl #y Ala Arg Leu Val Val305                 3 #10                 3 #15                 3 #20ctc gcc acc gcc acc cct ccg ggc tcc gtc ac#t gtg ccc cat ccc aac     1008Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Th #r Val Pro His Pro Asn                325   #               330   #               335atc gag gag gtt gct ctg tcc acc acc gga ga#g atc cct ttt tac ggc     1056Ile Glu Glu Val Ala Leu Ser Thr Thr Gly Gl #u Ile Pro Phe Tyr Gly            340       #           345       #           350aag gct atc ccc ctc gaa gta atc aag ggg gg#g aga cat ctc atc ttc     1104Lys Ala Ile Pro Leu Glu Val Ile Lys Gly Gl #y Arg His Leu Ile Phe        355           #       360           #       365tgt cat tca aag aag aag tgc gac gaa ctc gc#c gca aag ctg gtc gca     1152Cys His Ser Lys Lys Lys Cys Asp Glu Leu Al #a Ala Lys Leu Val Ala    370               #   375               #   380ttg ggc atc aat gcc gtg gcc tac tac cgc gg#t ctt gac gtg tcc gtc     1200Leu Gly Ile Asn Ala Val Ala Tyr Tyr Arg Gl #y Leu Asp Val Ser Val385                 3 #90                 3 #95                 4 #00atc ccg ccc atc ggc gat gtt gtc gtc gtg gc#a acc gat gcc ctc atg     1248Ile Pro Pro Ile Gly Asp Val Val Val Val Al #a Thr Asp Ala Leu Met                405   #               410   #               415acc ggc tat acg ggc gac ttc gac tcg gtg at#a gac tgc aat acg tgt     1296Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val Il #e Asp Cys Asn Thr Cys            420       #           425       #           430gtc acc cag aca gtc gat ttc agc ctt gac cc#t acc ttc acc att gag     1344Val Thr Gln Thr Val Asp Phe Ser Leu Asp Pr #o Thr Phe Thr Ile Glu        435           #       440           #       445aca atc acg ctc ccc caa gat gct gtc tcc cg#c act caa cgt cgg ggc     1392Thr Ile Thr Leu Pro Gln Asp Ala Val Ser Ar #g Thr Gln Arg Arg Gly    450               #   455               #   460agg act ggc agg ggg aag cca ggc atc tac ag#a ttt gtg gca ccg ggg     1440Arg Thr Gly Arg Gly Lys Pro Gly Ile Tyr Ar #g Phe Val Ala Pro Gly465                 4 #70                 4 #75                 4 #80gag cgc ccc tcc ggc atg ttc gac tcg tcc gt#c ctc tgt gag tgc tat     1488Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Va #l Leu Cys Glu Cys Tyr                485   #               490   #               495gac gca ggc tgt gct tgg tat gag ctc acg cc#c gcc gag act aca gtt     1536Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pr #o Ala Glu Thr Thr Val            500       #           505       #           510agg cta cga gcg tac atg aac acc ccg ggg ct#t ccc gtg tgc cag gac     1584Arg Leu Arg Ala Tyr Met Asn Thr Pro Gly Le #u Pro Val Cys Gln Asp        515           #       520           #       525cat ctt gaa ttt tgg gag ggc gtc ttt aca gg#c ctc act cat ata gat     1632His Leu Glu Phe Trp Glu Gly Val Phe Thr Gl #y Leu Thr His Ile Asp    530               #   535               #   540gcc cac ttt cta tcc cag aca aag cag agt gg#g gag aac ctt cct tac     1680Ala His Phe Leu Ser Gln Thr Lys Gln Ser Gl #y Glu Asn Leu Pro Tyr545                 5 #50                 5 #55                 5 #60ctg gta gcg tac caa gcc acc gtg tgc gct ag#g gct caa gcc cct ccc     1728Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Ar #g Ala Gln Ala Pro Pro                565   #               570   #               575cca tcg tgg gac cag atg tgg aag tgt ttg at#t cgc ctc aag ccc acc     1776Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Il #e Arg Leu Lys Pro Thr            580       #           585       #           590ctc cat ggg cca aca ccc ctg cta tac aga ct#g ggc gct gtt cag aat     1824Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Le #u Gly Ala Val Gln Asn        595           #       600           #       605gaa atc acc ctg acg cac cca gtc acc aaa ta#c atc atg aca tgc atg     1872Glu Ile Thr Leu Thr His Pro Val Thr Lys Ty #r Ile Met Thr Cys Met    610               #   615               #   620tcg gcc gac ctg gag gtc gtc acg agc acc tg#g gtg ctc gtt ggc ggc     1920Ser Ala Asp Leu Glu Val Val Thr Ser Thr Tr #p Val Leu Val Gly Gly625                 6 #30                 6 #35                 6 #40gtc ctg gct gct ttg gcc gcg tat tgc ctg tc#a aca ggc tgc gtg gtc     1968Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Se #r Thr Gly Cys Val Val                645   #               650   #               655ata gtg ggc agg gtc gtc ttg tcc ggg aag cc#g gca atc ata cct gac     2016Ile Val Gly Arg Val Val Leu Ser Gly Lys Pr #o Ala Ile Ile Pro Asp            660       #           665       #           670agg gaa gtc ctc tac cga gag ttc gat gag at #g gaa gag tgc             #2058 Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Me #t Glu Glu Cys        675           #       680           #       685<210> SEQ ID NO 2 <211> LENGTH: 686 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:      representative NS3/4a conformational an #tigen <400> SEQUENCE: 2Met Ala Pro Ile Thr Ala Tyr Ala Gln Gln Th #r Arg Gly Leu Leu Gly1               5    #                10   #                15Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Ly #s Asn Gln Val Glu Gly            20       #            25       #            30Glu Val Gln Ile Val Ser Thr Ala Ala Gln Th #r Phe Leu Ala Thr Cys        35           #        40           #        45Ile Asn Gly Val Cys Trp Thr Val Tyr His Gl #y Ala Gly Thr Arg Thr    50               #    55               #    60Ile Ala Ser Pro Lys Gly Pro Val Ile Gln Me #t Tyr Thr Asn Val Asp65                   #70                   #75                   #80Gln Asp Leu Val Gly Trp Pro Ala Pro Gln Gl #y Ser Arg Ser Leu Thr                85   #                90   #                95Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Le #u Val Thr Arg His Ala            100       #           105       #           110Asp Val Ile Pro Val Arg Arg Arg Gly Asp Se #r Arg Gly Ser Leu Leu        115           #       120           #       125Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly Se #r Ser Gly Gly Pro Leu    130               #   135               #   140Leu Cys Pro Ala Gly His Ala Val Gly Ile Ph #e Arg Ala Ala Val Cys145                 1 #50                 1 #55                 1 #60Thr Arg Gly Val Ala Lys Ala Val Asp Phe Il #e Pro Val Glu Asn Leu                165   #               170   #               175Glu Thr Thr Met Arg Ser Pro Val Phe Thr As #p Asn Ser Ser Pro Pro            180       #           185       #           190Val Val Pro Gln Ser Phe Gln Val Ala His Le #u His Ala Pro Thr Gly        195           #       200           #       205Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Ty #r Ala Ala Gln Gly Tyr    210               #   215               #   220Lys Val Leu Val Leu Asn Pro Ser Val Ala Al #a Thr Leu Gly Phe Gly225                 2 #30                 2 #35                 2 #40Ala Tyr Met Ser Lys Ala His Gly Ile Asp Pr #o Asn Ile Arg Thr Gly                245   #               250   #               255Val Arg Thr Ile Thr Thr Gly Ser Pro Ile Th #r Tyr Ser Thr Tyr Gly            260       #           265       #           270Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gl #y Ala Tyr Asp Ile Ile        275           #       280           #       285Ile Cys Asp Glu Cys His Ser Thr Asp Ala Th #r Ser Ile Leu Gly Ile    290               #   295               #   300Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gl #y Ala Arg Leu Val Val305                 3 #10                 3 #15                 3 #20Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Th #r Val Pro His Pro Asn                325   #               330   #               335Ile Glu Glu Val Ala Leu Ser Thr Thr Gly Gl #u Ile Pro Phe Tyr Gly            340       #           345       #           350Lys Ala Ile Pro Leu Glu Val Ile Lys Gly Gl #y Arg His Leu Ile Phe        355           #       360           #       365Cys His Ser Lys Lys Lys Cys Asp Glu Leu Al #a Ala Lys Leu Val Ala    370               #   375               #   380Leu Gly Ile Asn Ala Val Ala Tyr Tyr Arg Gl #y Leu Asp Val Ser Val385                 3 #90                 3 #95                 4 #00Ile Pro Pro Ile Gly Asp Val Val Val Val Al #a Thr Asp Ala Leu Met                405   #               410   #               415Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val Il #e Asp Cys Asn Thr Cys            420       #           425       #           430Val Thr Gln Thr Val Asp Phe Ser Leu Asp Pr #o Thr Phe Thr Ile Glu        435           #       440           #       445Thr Ile Thr Leu Pro Gln Asp Ala Val Ser Ar #g Thr Gln Arg Arg Gly    450               #   455               #   460Arg Thr Gly Arg Gly Lys Pro Gly Ile Tyr Ar #g Phe Val Ala Pro Gly465                 4 #70                 4 #75                 4 #80Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Va #l Leu Cys Glu Cys Tyr                485   #               490   #               495Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pr #o Ala Glu Thr Thr Val            500       #           505       #           510Arg Leu Arg Ala Tyr Met Asn Thr Pro Gly Le #u Pro Val Cys Gln Asp        515           #       520           #       525His Leu Glu Phe Trp Glu Gly Val Phe Thr Gl #y Leu Thr His Ile Asp    530               #   535               #   540Ala His Phe Leu Ser Gln Thr Lys Gln Ser Gl #y Glu Asn Leu Pro Tyr545                 5 #50                 5 #55                 5 #60Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Ar #g Ala Gln Ala Pro Pro                565   #               570   #               575Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Il #e Arg Leu Lys Pro Thr            580       #           585       #           590Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Le #u Gly Ala Val Gln Asn        595           #       600           #       605Glu Ile Thr Leu Thr His Pro Val Thr Lys Ty #r Ile Met Thr Cys Met    610               #   615               #   620Ser Ala Asp Leu Glu Val Val Thr Ser Thr Tr #p Val Leu Val Gly Gly625                 6 #30                 6 #35                 6 #40Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Se #r Thr Gly Cys Val Val                645   #               650   #               655Ile Val Gly Arg Val Val Leu Ser Gly Lys Pr #o Ala Ile Ile Pro Asp            660       #           665       #           670Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Me #t Glu Glu Cys        675           #       680           #       685<210> SEQ ID NO 3 <211> LENGTH: 3297 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:  MEFA 7.1<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(3297)<400> SEQUENCE: 3 atg gct aca aag gct gtt tgt gtt ttg aag gg#t gac ggc cca gtt caa       48Met Ala Thr Lys Ala Val Cys Val Leu Lys Gl #y Asp Gly Pro Val Gln1               5    #                10   #                15ggt att att aac ttc gag cag aag gaa agt aa#t gga cca gtg aag gtg       96Gly Ile Ile Asn Phe Glu Gln Lys Glu Ser As #n Gly Pro Val Lys Val            20       #            25       #            30tgg gga agc att aaa gga ctg act gaa ggc ct#g cat gga ttc cat gtt      144Trp Gly Ser Ile Lys Gly Leu Thr Glu Gly Le #u His Gly Phe His Val        35           #        40           #        45cat gag ttt gga gat aat aca gca ggc tgt ac#c agt gca ggt cct cac      192His Glu Phe Gly Asp Asn Thr Ala Gly Cys Th #r Ser Ala Gly Pro His    50               #    55               #    60ttt aat cct cta tcc aga aaa cac ggt ggg cc#a aag gat gaa gag agg      240Phe Asn Pro Leu Ser Arg Lys His Gly Gly Pr #o Lys Asp Glu Glu Arg65                   #70                   #75                   #80cat gtt gga gac ttg ggc aat gtg act gct ga#c aaa gat ggt gtg gcc      288His Val Gly Asp Leu Gly Asn Val Thr Ala As #p Lys Asp Gly Val Ala                85   #                90   #                95gat gtg tct att gaa gat tct gtg atc tca ct#c tca gga gac cat tgc      336Asp Val Ser Ile Glu Asp Ser Val Ile Ser Le #u Ser Gly Asp His Cys            100       #           105       #           110atc att ggc cgc aca ctg gtg gtc cat gaa aa#a gca gat gac ttg ggc      384Ile Ile Gly Arg Thr Leu Val Val His Glu Ly #s Ala Asp Asp Leu Gly        115           #       120           #       125aaa ggt gga aat gaa gaa agt aca aag aca gg#a aac gct gga agt cgt      432Lys Gly Gly Asn Glu Glu Ser Thr Lys Thr Gl #y Asn Ala Gly Ser Arg    130               #   135               #   140ttg gct tgt ggt gta att ggg atc gcc cag aa#t ttg aat tct ggt tgc      480Leu Ala Cys Gly Val Ile Gly Ile Ala Gln As #n Leu Asn Ser Gly Cys145                 1 #50                 1 #55                 1 #60aat tgc tct atc tat ccc ggc cat ata acg gg#t cac cgc atg gca tgg      528Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gl #y His Arg Met Ala Trp                165   #               170   #               175aag ctt ggt tcc gcc gcc aga act acc tcg gg#c ttt gtc tcc ttg ttc      576Lys Leu Gly Ser Ala Ala Arg Thr Thr Ser Gl #y Phe Val Ser Leu Phe            180       #           185       #           190gcc cca ggt gcc aaa caa aac gaa act cac gt#c acg gga ggc gca gcc      624Ala Pro Gly Ala Lys Gln Asn Glu Thr His Va #l Thr Gly Gly Ala Ala        195           #       200           #       205gcc cga act acg tct ggg ttg acc tct ttg tt#c tcc cca ggt gcc agc      672Ala Arg Thr Thr Ser Gly Leu Thr Ser Leu Ph #e Ser Pro Gly Ala Ser    210               #   215               #   220caa aac att caa ttg att gtc gac ttt atc cc#t gtg gag aac cta gag      720Gln Asn Ile Gln Leu Ile Val Asp Phe Ile Pr #o Val Glu Asn Leu Glu225                 2 #30                 2 #35                 2 #40aca acc atg cga tct ccg gtg ttc acg gat aa#c tcc tct cca cca gta      768Thr Thr Met Arg Ser Pro Val Phe Thr Asp As #n Ser Ser Pro Pro Val                245   #               250   #               255gtg ccc cag agc ttc cag gtg gct cac ctc ca#t gct ccc aca ggc agc      816Val Pro Gln Ser Phe Gln Val Ala His Leu Hi #s Ala Pro Thr Gly Ser            260       #           265       #           270ggc aaa agc acc aag gtc ccg gct gca tat gc#a gct cag ggc tat aag      864Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Al #a Ala Gln Gly Tyr Lys        275           #       280           #       285gtg cta gta ctc aac ccc tct gtt gct gca ac#a ctg ggc ttt ggt gct      912Val Leu Val Leu Asn Pro Ser Val Ala Ala Th #r Leu Gly Phe Gly Ala    290               #   295               #   300tac atg tcc aag gct cat ggg atc gat cct aa#c atc agg acc ggg gtg      960Tyr Met Ser Lys Ala His Gly Ile Asp Pro As #n Ile Arg Thr Gly Val305                 3 #10                 3 #15                 3 #20aga aca att acc act ggc agc ccc atc acg ta#c tcc acc tac ggc aag     1008Arg Thr Ile Thr Thr Gly Ser Pro Ile Thr Ty #r Ser Thr Tyr Gly Lys                325   #               330   #               335ttc ctt gcc gac ggc ggg tgc tcg ggg ggc gc#t tat gac ata ata att     1056Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Al #a Tyr Asp Ile Ile Ile            340       #           345       #           350tgt gac gag tgc cac tcc acg gat gcc aca tc#c atc ttg ggc att ggc     1104Cys Asp Glu Cys His Ser Thr Asp Ala Thr Se #r Ile Leu Gly Ile Gly        355           #       360           #       365act gtc ctt gac caa gca gag act gcg ggg gc#g aga ctg gtt gtg ctc     1152Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Al #a Arg Leu Val Val Leu    370               #   375               #   380gcc acc gcc acc cct ccg ggc tcc gtc act gt#g ccc cat ccc aac atc     1200Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Va #l Pro His Pro Asn Ile385                 3 #90                 3 #95                 4 #00gag gag gtt gct ctg tcc acc acc gga gag at#c cct ttt tac ggc aag     1248Glu Glu Val Ala Leu Ser Thr Thr Gly Glu Il #e Pro Phe Tyr Gly Lys                405   #               410   #               415gct atc ccc ctc gaa gta atc aag ggg ggg ag#a cat ctc atc ttc tgt     1296Ala Ile Pro Leu Glu Val Ile Lys Gly Gly Ar #g His Leu Ile Phe Cys            420       #           425       #           430cat tca aag aag aag tgc gac gaa ctc gcc gc#a aag ctg gtc gca ttg     1344His Ser Lys Lys Lys Cys Asp Glu Leu Ala Al #a Lys Leu Val Ala Leu        435           #       440           #       445ggc atc aat gcc gtg gcc tac tac cgc ggt ct#t gac gtg tcc gtc atc     1392Gly Ile Asn Ala Val Ala Tyr Tyr Arg Gly Le #u Asp Val Ser Val Ile    450               #   455               #   460ccg acc agc ggc gat gtt gtc gtc gtg gca ac#c gat gcc ctc atg acc     1440Pro Thr Ser Gly Asp Val Val Val Val Ala Th #r Asp Ala Leu Met Thr465                 4 #70                 4 #75                 4 #80ggc tat acc ggc gac ttc gac tcg gtg ata ga#c tgc aat acg tgt gtc     1488Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile As #p Cys Asn Thr Cys Val                485   #               490   #               495acc cag aca gtc gat ttc agc ctt gac cct ac#c ttc acc att gag aca     1536Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Th #r Phe Thr Ile Glu Thr            500       #           505       #           510atc acg ctc ccc caa gat gct gtc tcc cgc ac#t caa cgt cgg ggc agg     1584Ile Thr Leu Pro Gln Asp Ala Val Ser Arg Th #r Gln Arg Arg Gly Arg        515           #       520           #       525act ggc agg ggg aag cca ggc atc tac aga tt#t gtg gca ccg ggg gag     1632Thr Gly Arg Gly Lys Pro Gly Ile Tyr Arg Ph #e Val Ala Pro Gly Glu    530               #   535               #   540cgc ccc tcc ggc atg ttc gac tcg tcc gtc ct#c tgt gag tgc tat gac     1680Arg Pro Ser Gly Met Phe Asp Ser Ser Val Le #u Cys Glu Cys Tyr Asp545                 5 #50                 5 #55                 5 #60gca ggc tgt gct tgg tat gag ctc acg ccc gc#c gag act aca gtt agg     1728Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Al #a Glu Thr Thr Val Arg                565   #               570   #               575cta cga gcg tac atg aac acc ccg ggg ctt cc#c gtg tgc cag gac cat     1776Leu Arg Ala Tyr Met Asn Thr Pro Gly Leu Pr #o Val Cys Gln Asp His            580       #           585       #           590ctt gaa ttt tgg gag ggc gtc ttt aca ggc ct#c act cat ata gat gcc     1824Leu Glu Phe Trp Glu Gly Val Phe Thr Gly Le #u Thr His Ile Asp Ala        595           #       600           #       605cac ttt cta tcc cag aca aag cag agt ggg ga#g aac ctt cct tac ctg     1872His Phe Leu Ser Gln Thr Lys Gln Ser Gly Gl #u Asn Leu Pro Tyr Leu    610               #   615               #   620gta gcg tac caa gcc acc gtg tgc gct agg gc#t caa gcc cct ccc cca     1920Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Al #a Gln Ala Pro Pro Pro625                 6 #30                 6 #35                 6 #40tcg tgg gac cag atg tgg aag tgt ttg att cg#c ctc aag ccc acc ctc     1968Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Ar #g Leu Lys Pro Thr Leu                645   #               650   #               655cat ggg cca aca ccc ctg cta tac aga ctg gg#c gct gtt cag aat gaa     2016His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gl #y Ala Val Gln Asn Glu            660       #           665       #           670atc acc ctg acg cac cca gtc acc aaa tac at#c atg aca tgc atg tcg     2064Ile Thr Leu Thr His Pro Val Thr Lys Tyr Il #e Met Thr Cys Met Ser        675           #       680           #       685gcc gac ctg gag gtc gtc acg agc gca tgc tc#c ggg aag ccg gca atc     2112Ala Asp Leu Glu Val Val Thr Ser Ala Cys Se #r Gly Lys Pro Ala Ile    690               #   695               #   700ata cct gac agg gaa gtc ctc tac cga gag tt#c gat gag atg gaa gag     2160Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu Ph #e Asp Glu Met Glu Glu705                 7 #10                 7 #15                 7 #20tgc tct cag cac tta ccg tac atc gag caa gg#g atg atg ctc gcc gag     2208Cys Ser Gln His Leu Pro Tyr Ile Glu Gln Gl #y Met Met Leu Ala Glu                725   #               730   #               735cag ttc aag cag aag gcc ctc ggc ctc tcg cg#a ggg ggc aag ccg gca     2256Gln Phe Lys Gln Lys Ala Leu Gly Leu Ser Ar #g Gly Gly Lys Pro Ala            740       #           745       #           750atc gtt cca gac aaa gag gtg ttg tat caa ca#a tac gat gag atg gaa     2304Ile Val Pro Asp Lys Glu Val Leu Tyr Gln Gl #n Tyr Asp Glu Met Glu        755           #       760           #       765gag tgc tca caa gct gcc cca tat atc gaa ca#a gct cag gta ata gct     2352Glu Cys Ser Gln Ala Ala Pro Tyr Ile Glu Gl #n Ala Gln Val Ile Ala    770               #   775               #   780cac cag ttc aag gaa aaa gtc ctt gga ttg at#c gat aat gat caa gtg     2400His Gln Phe Lys Glu Lys Val Leu Gly Leu Il #e Asp Asn Asp Gln Val785                 7 #90                 7 #95                 8 #00gtt gtg act cct gac aaa gaa atc tta tat ga#g gcc ttt gat gag atg     2448Val Val Thr Pro Asp Lys Glu Ile Leu Tyr Gl #u Ala Phe Asp Glu Met                805   #               810   #               815gaa gaa tgc gcc tcc aaa gcc gcc ctc att ga#g gaa ggg cag cgg atg     2496Glu Glu Cys Ala Ser Lys Ala Ala Leu Ile Gl #u Glu Gly Gln Arg Met            820       #           825       #           830gcg gag atg ctc aag tct aag ata caa ggc ct#c ctc ggg ata ctg cgc     2544Ala Glu Met Leu Lys Ser Lys Ile Gln Gly Le #u Leu Gly Ile Leu Arg        835           #       840           #       845cgg cac gtt ggt cct ggc gag ggg gca gtg ca#g tgg atg aac cgg ctg     2592Arg His Val Gly Pro Gly Glu Gly Ala Val Gl #n Trp Met Asn Arg Leu    850               #   855               #   860ata gcc ttc gcc tcc aga ggg aac cat gtt tc#c ccc acg cac tac gtt     2640Ile Ala Phe Ala Ser Arg Gly Asn His Val Se #r Pro Thr His Tyr Val865                 8 #70                 8 #75                 8 #80ccg tct aga tcc cgg aga ttc gcc cag gcc ct#g ccc gtt tgg gcg cgg     2688Pro Ser Arg Ser Arg Arg Phe Ala Gln Ala Le #u Pro Val Trp Ala Arg                885   #               890   #               895ccg gac tat aac ccc ccg cta gtg gag acg tg#g aaa aag ccc gac tac     2736Pro Asp Tyr Asn Pro Pro Leu Val Glu Thr Tr #p Lys Lys Pro Asp Tyr            900       #           905       #           910gaa cca cct gtg gtc cac ggc aga tct tct cg#g aga ttc gcc cag gcc     2784Glu Pro Pro Val Val His Gly Arg Ser Ser Ar #g Arg Phe Ala Gln Ala        915           #       920           #       925ctg ccc gtt tgg gcg cgg ccg gac tat aac cc#c ccg cta gtg gag acg     2832Leu Pro Val Trp Ala Arg Pro Asp Tyr Asn Pr #o Pro Leu Val Glu Thr    930               #   935               #   940tgg aaa aag ccc gac tac gaa cca cct gtg gt#c cat ggc aga aag acc     2880Trp Lys Lys Pro Asp Tyr Glu Pro Pro Val Va #l His Gly Arg Lys Thr945                 9 #50                 9 #55                 9 #60aaa cgt aac acc aac cgg cgg ccg cag gac gt#c aag ttc ccg ggt ggc     2928Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Va #l Lys Phe Pro Gly Gly                965   #               970   #               975ggt cag atc gtt ggt cgc agg ggc cct cct at#c ccc aag gct cgt cgg     2976Gly Gln Ile Val Gly Arg Arg Gly Pro Pro Il #e Pro Lys Ala Arg Arg            980       #           985       #           990ccc gag ggc agg acc tgg gct cag ccc ggt ta#c cct tgg ccc ctc tat     3024Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly Ty #r Pro Trp Pro Leu Tyr        995           #       1000           #      1005ggc aat aag gac aga cgg tct aca ggt aag tc#c tgg ggt aag cca ggg     3072Gly Asn Lys Asp Arg Arg Ser Thr Gly Lys Se #r Trp Gly Lys Pro Gly    1010              #   1015               #  1020tac cct tgg cca aga aag acc aaa cgt aac ac#c aac cga cgg ccg cag     3120Tyr Pro Trp Pro Arg Lys Thr Lys Arg Asn Th #r Asn Arg Arg Pro Gln1025                1030 #                1035  #               1040gac gtc aag ttc ccg ggt ggc ggt cag atc gt#t ggt cgc agg ggc cct     3168Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Va #l Gly Arg Arg Gly Pro                1045  #               1050   #              1055cct atc ccc aag gct cgt cgg ccc gag ggc ag#g acc tgg gct cag ccc     3216Pro Ile Pro Lys Ala Arg Arg Pro Glu Gly Ar #g Thr Trp Ala Gln Pro            1060      #           1065       #          1070ggt tac cct tgg ccc ctc tat ggc aat aag ga#c aga cgg tct acc ggt     3264Gly Tyr Pro Trp Pro Leu Tyr Gly Asn Lys As #p Arg Arg Ser Thr Gly        1075          #       1080           #      1085aag tcc tgg ggt aag cca ggg tat cct tgg cc #c                  #       3297 Lys Ser Trp Gly Lys Pro Gly Tyr Pro Trp Pr #o    1090              #   1095 <210> SEQ ID NO 4 <211> LENGTH: 1099<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:  MEFA 7.1<400> SEQUENCE: 4 Met Ala Thr Lys Ala Val Cys Val Leu Lys Gl#y Asp Gly Pro Val Gln 1               5    #                10  #                15 Gly Ile Ile Asn Phe Glu Gln Lys Glu Ser As#n Gly Pro Val Lys Val             20       #            25      #            30 Trp Gly Ser Ile Lys Gly Leu Thr Glu Gly Le#u His Gly Phe His Val         35           #        40          #        45 His Glu Phe Gly Asp Asn Thr Ala Gly Cys Th#r Ser Ala Gly Pro His     50               #    55              #    60 Phe Asn Pro Leu Ser Arg Lys His Gly Gly Pr#o Lys Asp Glu Glu Arg 65                   #70                  #75                   #80 His Val Gly Asp Leu Gly Asn Val Thr Ala As#p Lys Asp Gly Val Ala                 85   #                90  #                95 Asp Val Ser Ile Glu Asp Ser Val Ile Ser Le#u Ser Gly Asp His Cys             100       #           105      #           110 Ile Ile Gly Arg Thr Leu Val Val His Glu Ly#s Ala Asp Asp Leu Gly         115           #       120          #       125 Lys Gly Gly Asn Glu Glu Ser Thr Lys Thr Gl#y Asn Ala Gly Ser Arg     130               #   135              #   140 Leu Ala Cys Gly Val Ile Gly Ile Ala Gln As#n Leu Asn Ser Gly Cys 145                 1 #50                 1#55                 1 #60 Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gl#y His Arg Met Ala Trp                 165   #               170  #               175 Lys Leu Gly Ser Ala Ala Arg Thr Thr Ser Gl#y Phe Val Ser Leu Phe             180       #           185      #           190 Ala Pro Gly Ala Lys Gln Asn Glu Thr His Va#l Thr Gly Gly Ala Ala         195           #       200          #       205 Ala Arg Thr Thr Ser Gly Leu Thr Ser Leu Ph#e Ser Pro Gly Ala Ser     210               #   215              #   220 Gln Asn Ile Gln Leu Ile Val Asp Phe Ile Pr#o Val Glu Asn Leu Glu 225                 2 #30                 2#35                 2 #40 Thr Thr Met Arg Ser Pro Val Phe Thr Asp As#n Ser Ser Pro Pro Val                 245   #               250  #               255 Val Pro Gln Ser Phe Gln Val Ala His Leu Hi#s Ala Pro Thr Gly Ser             260       #           265      #           270 Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Al#a Ala Gln Gly Tyr Lys         275           #       280          #       285 Val Leu Val Leu Asn Pro Ser Val Ala Ala Th#r Leu Gly Phe Gly Ala     290               #   295              #   300 Tyr Met Ser Lys Ala His Gly Ile Asp Pro As#n Ile Arg Thr Gly Val 305                 3 #10                 3#15                 3 #20 Arg Thr Ile Thr Thr Gly Ser Pro Ile Thr Ty#r Ser Thr Tyr Gly Lys                 325   #               330  #               335 Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Al#a Tyr Asp Ile Ile Ile             340       #           345      #           350 Cys Asp Glu Cys His Ser Thr Asp Ala Thr Se#r Ile Leu Gly Ile Gly         355           #       360          #       365 Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Al#a Arg Leu Val Val Leu     370               #   375              #   380 Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Va#l Pro His Pro Asn Ile 385                 3 #90                 3#95                 4 #00 Glu Glu Val Ala Leu Ser Thr Thr Gly Glu Il#e Pro Phe Tyr Gly Lys                 405   #               410  #               415 Ala Ile Pro Leu Glu Val Ile Lys Gly Gly Ar#g His Leu Ile Phe Cys             420       #           425      #           430 His Ser Lys Lys Lys Cys Asp Glu Leu Ala Al#a Lys Leu Val Ala Leu         435           #       440          #       445 Gly Ile Asn Ala Val Ala Tyr Tyr Arg Gly Le#u Asp Val Ser Val Ile     450               #   455              #   460 Pro Thr Ser Gly Asp Val Val Val Val Ala Th#r Asp Ala Leu Met Thr 465                 4 #70                 4#75                 4 #80 Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile As#p Cys Asn Thr Cys Val                 485   #               490  #               495 Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Th#r Phe Thr Ile Glu Thr             500       #           505      #           510 Ile Thr Leu Pro Gln Asp Ala Val Ser Arg Th#r Gln Arg Arg Gly Arg         515           #       520          #       525 Thr Gly Arg Gly Lys Pro Gly Ile Tyr Arg Ph#e Val Ala Pro Gly Glu     530               #   535              #   540 Arg Pro Ser Gly Met Phe Asp Ser Ser Val Le#u Cys Glu Cys Tyr Asp 545                 5 #50                 5#55                 5 #60 Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Al#a Glu Thr Thr Val Arg                 565   #               570  #               575 Leu Arg Ala Tyr Met Asn Thr Pro Gly Leu Pr#o Val Cys Gln Asp His             580       #           585      #           590 Leu Glu Phe Trp Glu Gly Val Phe Thr Gly Le#u Thr His Ile Asp Ala         595           #       600          #       605 His Phe Leu Ser Gln Thr Lys Gln Ser Gly Gl#u Asn Leu Pro Tyr Leu     610               #   615              #   620 Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Al#a Gln Ala Pro Pro Pro 625                 6 #30                 6#35                 6 #40 Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Ar#g Leu Lys Pro Thr Leu                 645   #               650  #               655 His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gl#y Ala Val Gln Asn Glu             660       #           665      #           670 Ile Thr Leu Thr His Pro Val Thr Lys Tyr Il#e Met Thr Cys Met Ser         675           #       680          #       685 Ala Asp Leu Glu Val Val Thr Ser Ala Cys Se#r Gly Lys Pro Ala Ile     690               #   695              #   700 Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu Ph#e Asp Glu Met Glu Glu 705                 7 #10                 7#15                 7 #20 Cys Ser Gln His Leu Pro Tyr Ile Glu Gln Gl#y Met Met Leu Ala Glu                 725   #               730  #               735 Gln Phe Lys Gln Lys Ala Leu Gly Leu Ser Ar#g Gly Gly Lys Pro Ala             740       #           745      #           750 Ile Val Pro Asp Lys Glu Val Leu Tyr Gln Gl#n Tyr Asp Glu Met Glu         755           #       760          #       765 Glu Cys Ser Gln Ala Ala Pro Tyr Ile Glu Gl#n Ala Gln Val Ile Ala     770               #   775              #   780 His Gln Phe Lys Glu Lys Val Leu Gly Leu Il#e Asp Asn Asp Gln Val 785                 7 #90                 7#95                 8 #00 Val Val Thr Pro Asp Lys Glu Ile Leu Tyr Gl#u Ala Phe Asp Glu Met                 805   #               810  #               815 Glu Glu Cys Ala Ser Lys Ala Ala Leu Ile Gl#u Glu Gly Gln Arg Met             820       #           825      #           830 Ala Glu Met Leu Lys Ser Lys Ile Gln Gly Le#u Leu Gly Ile Leu Arg         835           #       840          #       845 Arg His Val Gly Pro Gly Glu Gly Ala Val Gl#n Trp Met Asn Arg Leu     850               #   855              #   860 Ile Ala Phe Ala Ser Arg Gly Asn His Val Se#r Pro Thr His Tyr Val 865                 8 #70                 8#75                 8 #80 Pro Ser Arg Ser Arg Arg Phe Ala Gln Ala Le#u Pro Val Trp Ala Arg                 885   #               890  #               895 Pro Asp Tyr Asn Pro Pro Leu Val Glu Thr Tr#p Lys Lys Pro Asp Tyr             900       #           905      #           910 Glu Pro Pro Val Val His Gly Arg Ser Ser Ar#g Arg Phe Ala Gln Ala         915           #       920          #       925 Leu Pro Val Trp Ala Arg Pro Asp Tyr Asn Pr#o Pro Leu Val Glu Thr     930               #   935              #   940 Trp Lys Lys Pro Asp Tyr Glu Pro Pro Val Va#l His Gly Arg Lys Thr 945                 9 #50                 9#55                 9 #60 Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Va#l Lys Phe Pro Gly Gly                 965   #               970  #               975 Gly Gln Ile Val Gly Arg Arg Gly Pro Pro Il#e Pro Lys Ala Arg Arg             980       #           985      #           990 Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly Ty#r Pro Trp Pro Leu Tyr         995           #       1000          #      1005 Gly Asn Lys Asp Arg Arg Ser Thr Gly Lys Se#r Trp Gly Lys Pro Gly     1010              #   1015              #  1020 Tyr Pro Trp Pro Arg Lys Thr Lys Arg Asn Th#r Asn Arg Arg Pro Gln 1025                1030 #                1035 #               1040 Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Va#l Gly Arg Arg Gly Pro                 1045  #               1050  #              1055 Pro Ile Pro Lys Ala Arg Arg Pro Glu Gly Ar#g Thr Trp Ala Gln Pro             1060      #           1065      #          1070 Gly Tyr Pro Trp Pro Leu Tyr Gly Asn Lys As#p Arg Arg Ser Thr Gly         1075          #       1080          #      1085 Lys Ser Trp Gly Lys Pro Gly Tyr Pro Trp Pr #o    1090              #   1095 <210> SEQ ID NO 5 <211> LENGTH: 21<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial #Sequence:  consensus       sequence <400> SEQUENCE: 5Gly Ser Ala Ala Arg Thr Thr Ser Gly Phe Va #l Ser Leu Phe Ala Pro1               5    #                10   #                15Gly Ala Lys Gln Asn             20 <210> SEQ ID NO 6 <211> LENGTH: 10<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:  ligated      DNA piece <400> SEQUENCE: 6 acaaaacaaa                #                   #                   #        10 <210> SEQ ID NO 7<211> LENGTH: 23 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence:  NS4A       peptide <400> SEQUENCE: 7Lys Lys Gly Ser Val Val Ile Val Gly Arg Il #e Val Leu Ser Gly Lys1               5    #                10   #                15Pro Ala Ile Ile Pro Lys Lys             20

What is claimed is:
 1. A multiple epitope fusion antigen comprising theamino acid sequence of SEQ ID NO:4, or an amino acid sequence with atleast 80% sequence identity thereto which reacts specifically withanti-HCV antibodies that specifically react with SEQ ID NO:4 present ina biological sample from an HCV-infected individual.
 2. The multipleepitope fusion antigen of claim 1, wherein said multiple epitope fusionantigen comprises the amino acid sequence of SEQ ID NO:4, or an aminoacid sequence with at least 90% sequence identity thereto which reactsspecifically with anti-HCV antibodies that specifically react with SEQID NO:4 present in a biological sample from an HCV-infected individual.3. The multiple epitope fusion antigen of claim 1, wherein said multipleepitope fusion antigen comprises the amino acid sequence of SEQ ID NO:4,or an amino acid sequence with at least 98% sequence identity theretowhich reacts specifically with anti-HCV antibodies that specificallyreact with SEQ ID NO:4 present in a biological sample from anHCV-infected individual.
 4. The multiple epitope fusion antigen of claim1, wherein said multiple epitope fusion antigen consists of the aminoacid sequence of SEQ ID NO:4.